Fgf-9 variants and methods of use thereof

ABSTRACT

A method of treating an individual (i) having abnormal bone; or (ii) afflicted with a disease or disorder related to normal or abnormal FGF receptors or a skeletal disorder; or (iii) having dysplasic bone. The method includes administering to the individual a pharmaceutical composition comprising a therapeutically effective amount of a fibroblast growth factor 9 (FGF-9) variant comprising at least one amino acid substitution in the beta 8-beta 9 loop, wherein said FGF-9 variant incorporates one of the amino acid sequences set forth in SEQ ID NO: 11, 13, 14, 15, 16 or 17.

FIELD OF THE INVENTION

The present invention concerns mutants and variants of fibroblast growthfactors (FGFs) with improved properties, and provides FGF polypeptides,pharmaceutical compositions comprising these variants and methods foruse thereof.

BACKGROUND OF THE INVENTION

Fibroblast Growth Factors and their Receptors

Fibroblast growth factors (FGFs) comprise a large family ofevolutionarily conserved polypeptides involved in a variety ofbiological processes including morphogenesis, angiogenesis, and tissueremodeling as well as in the pathogenesis of numerous diseases (reviewedin Ornitz, Bioessays 22, 108, 2000). The various members of this familystimulate the proliferation of a wide spectrum of cells, ranging frommesenchymal to epithelial and neuroectodermal origin in vitro and invivo. FGFs are expressed in a strict temporal and spatial pattern duringdevelopment and have important roles in patterning and limb formation(reviewed in Ornitz, Bioessays 22, 108, 2000).

FGFs are powerful mitogens and are critical in the regulation of manybiological processes including angiogenesis, vasculogenesis, woundhealing, limb formation, tumorigenesis and cell survival. The biologicalresponse of cells to FGF is mediated through specific, high affinity (Kd20-500 pM) cell surface receptors that possess intrinsic tyrosine kinaseactivity and are phosphorylated upon binding of FGF (Coughlin et al. JBiol. Chem. 263, 988, 1988). Five distinct Fibroblast Growth FactorReceptors (FGFRs) have been identified, FGFR1-4 aretransmembrane-protein kinases while FGFR5 appears to be a solublereceptor. The FGFR extracellular domain consists of threeimmunoglobulin-like (Ig-like) domains (D1, D2 and D3), a heparin bindingdomain and an acidic box. Alternative splicing of the FGFR mRNAsgenerates different receptor variants, including the FGFR3IIIb andFGFR3IIIc forms, each having unique ligand specificity.

Another critically functional component in receptor activation is thebinding to proteoglycans such as heparan sulfate. FGFs fail to bind andactivate FGF receptors in cells deprived of endogenous heparan sulfate.Different models have been proposed to explain the role of heparansulfate proteoglycans (HSPG) in FGF signaling, including the formationof a functional tertiary complex between FGF, FGFR and an HSPG (Yayon etal., Cell 64, 841, 1991; Faham et al, Curr. Opin. Struct. Biol. 8: 578,1998).

Bone Development

The process of bone formation is initiated by endochondral ossificationand intramembranous ossification. Endochondral ossification is thefundamental mechanism for longitudinal bone formation whereby cartilageis replaced by bone. It requires the sequential formation anddegradation of cartilaginous structures in the growth plates that serveas templates for the developing bones. During intramembranousossification, bone is formed directly in the connective tissues. Bothprocesses require the infiltration of osteoblasts and subsequent matrixdeposition.

The signaling pathway triggered by activation of FGFRs has been shown tobe involved in several stages of limb and bone development. Other majorregulators of bone growth include natriuretic peptides (NP), bonemorphogenetic proteins (BMP), growth hormone (GH), insulin-like growthfactors (IGF), glucocorticoids (GC), thyroid hormone (TH), parathyroidhormone (PTH), PTH related peptide (PTHrP) and Vitamin D.

FGFRs and Disease

A number of birth defects affecting the skeleton are associated withmutations in the genes encoding FGF receptors, specifically Crouzon,Pfeiffer, Jackson-Weiss, Apert and Beare-Stevenson syndromes (Kan, etal., Am J Hum Genet. 70, 472, 2002). Mutations in FGFR3 are responsiblefor achondroplasia, the most common form of human genetic dwarfism(reviewed in Vajo et al., Endocr. Rev. 21, 23, 2000). Specifically, theoutcome of the achondroplasia mutation is a stabilized, constitutivelyactivated FGFR3 leading to restricted chondrocyte maturation in thegrowth plate of long bones and abnormally shortened bones.

The FGFRs have been implicated in certain malignancies and proliferativediseases. FGFR3 is the most frequently mutated oncogene in transitionalcell carcinoma (TCC) of the bladder where it is mutated in more than 30%of the cases (Cappellen et al., Nature Genet. 23, 18, 1999). Dvorakovaet al. (Br. J. Haematol. 113, 832, 2001) have shown that the FGFR3IIIcisoform is over expressed in the white blood cells of chronic myeloidleukemia (CML) patients. Yee et al. (J. Natl. Cancer 92, 1848, 2000)identified a mutation in FGFR3 linked to cervical carcinoma. Recently,FGFR4 was shown to be associated with pituitary tumors (Ezzat, et al, J.Clin. Invest. 109, 69, 2002) and breast cancer progression (Bange, etal., Cancer Res. 62, 840, 2002).

In contrast, FGFs and their analogs have been shown to be useful fortreating indications including wounds (U.S. Pat. Nos. 4,950,483,5,859,208 and 6,294,359), myocardial infarction (U.S. Pat. Nos.4,296,100 and 4,378,347), skeletal disorders (U.S. Pat. Nos. 5,614,496and 5,656,598) and for remodeling cardiac tissue (U.S. Pat. No.6,352,971).

Receptor Specificity

In light of the large number of FGFs and FGF receptor variants, a majorquestion regarding FGF function is their receptor specificity. All FGFRstested so far bind FGF-1 (acidic FGF, aFGF) with moderate to highaffinity, demonstrating an apparent redundancy in the FGF system. Incontrast to FGFR1 and FGFR2, the third receptor subtype, FGFR3 was foundto bind to FGF-8, FGF-17 and FGF-18 with high affinity and to FGF-9 withimproved selectivity. Specificity may also be achieved by specificproteoglycans expressed in different tissues (Ornitz, Bioessays, 22,108, 2000). Site-directed mutagenesis and X-ray crystallography wereused to study the basis of specificity of FGFs to their receptors(Plotnikov et al., Cell 98, 641, 1999; Plotnikov et al., Cell 101, 413,2000; Stauber et al., PNAS 97, 49, 2000; Pellegrini et al., Nature, 407,1029, 2000; Schlessinger et al., Mol Cell, 6, 43, 2000).

FGF Variants

All members of the FGF family share a homology core domain of about 120amino acids, 28 aa residues are highly conserved and six are identical.Structural studies on several FGFs identified 12 antiparallel β strandseach one adjacent to β-loops comprising the core region, conservedthroughout the family. The core domain comprises the primary FGFR andheparin binding sites. Receptor binding regions are distinct fromheparin binding regions (reviewed in Ornitz and Itoh, Gen. Biol. 2,3005.1, 2001).

Attempts have been made to achieve altered FGF receptor specificity bydeletions or truncations of its ligands, by means of mutationsintroduced at certain locations within the gene encoding for theproteins. Copending PCT application WO 02/36732 discloses FGF variantshaving at least one mutation in the β8-β9 loop, having increasedreceptor specificity to one receptor subtype compared to thecorresponding wild type FGF.

Several investigators have demonstrated FGF mutants and variantsaffecting receptor and heparin binding. Kuroda et al., (Bone, 25, 431,1999) demonstrated that a full-length FGF-4 polypeptide and a shortenedversion containing 134 amino acid residues exhibit comparable cellularproliferation and effect on increase of bone density. The shortest formof FGF-4 tested, containing only 111 amino acid residues, exhibitedlimited growth stimulatory activity.

U.S. Pat. No. 5,998,170 discloses a biologically active FGF-16 moleculehaving from one to thirty-four amino acids deleted from the N-terminusor from one to eighteen amino acids deleted from the C-terminus.

U.S. Pat. No. 5,512,460 discloses an FGF-9 (glia activating factor, GAF)molecule comprising N-terminus and C-terminus truncations of 53 aa and13 aa, respectively. U.S. Pat. No. 5,571,895 discloses a 54 aa deletionfrom the N-terminus of the protein yielding a 154 aa protein retainingits biological activity.

Basic FGF, also known as FGF-2, bFGF, prostatin and heparin bindinggrowth factor 2, is highly conserved among species and has been shown tostimulate the proliferation of a wide variety of cell types. Thesequence of FGF-2 has been disclosed U.S. Pat. Nos. 4,994,559;5,155,214; 5,439,818 and 5,604,293. Human FGF-2 is expressed in severalforms, a 210 aa precursor, a 155 aa form, a 146 aa N-terminal truncatedform and several others (reviewed in Okada-Ban et al., Int J BiochemCell Biol, 32, 263, 2000).

FGF-2 has been modified to alter biological properties and bindingspecificity. U.S. Pat. No. 5,491,220 discloses structural analoguescomprising substitution of the β9-β10 loop having altered biologicalproperties and binding specificity. Seno et al. (Eur. J. Biochem. 188,239, 1990) demonstrated that removal of the C-terminus, not theN-terminus, affects FGF-2 affinity to heparin.

Bailly et al. (FASEB J, 14, 333, 2000) show that FGF-2 mitogenic anddifferentiation activities may be dissociated by a point mutation inSer117 (S117A).

Human FGF-2 superagonists have been designed with substitutions ateither one or more of the following amino acids: glutamate 89, aspartate101 and/or leucine 137 (U.S. Pat. No. 6,274,712; note that the aanumbering is according to the 146 aa form of FGF-2 disclosed in Zhang etal, PNAS 88: 3446, 1991). U.S. Pat. No. 6,294,359 discloses agonist andantagonist analogs of FGF-2 that comprise amino acid substitutions atcertain heparin and receptor binding domains but does not teach receptorspecificity changes.

U.S. Pat. Nos. 5,302,702 and 5,310,883 disclose a recombinant FGF-2variant, having the alanine of position 3 and the serine of position 5replaced with glutamic acid, exhibiting increased yields.

The use of FGFs and FGF fragments for targeting cytotoxic agents hasbeen disclosed in WO 01/39788 and U.S. Pat. Nos. 5,191,067; 5,576,288;5,679,637. A mitogenically active FGF molecule provides a route forintroducing the selected agents into the cell.

The extensive efforts made to produce truncation, deletion and pointmutation variants in FGF have resulted in changes in affinity to thereceptors but not in significant alterations in receptor specificity.Thus, there is an unmet need for highly active and selective ligands forthe various types of FGF receptors, useful in selective stimulation orinhibition of these receptors, thereby addressing the clinicalmanifestations associated with the above-mentioned mutations, andmodulating various biological functions.

It is to be explicitly understood that known variants of FGFs areexcluded from the present invention.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide variants of membersof the FGF family of growth factors with improved receptor specificityand/or affinity and biological activity having a mutation in a majorvariable protein domain.

It is another object of the present invention to provide FGF variantshaving improved selectivity for receptor activation.

It is a still another object of the present invention to providevariants of members of the FGF family wherein certain specifictruncations of the carboxy and/or amino termini renders themadvantageous in that they are more stable, with improved receptorspecificity, and/or better targeting agents.

It is a further object of the present invention to provide apharmaceutical composition comprising variants useful in effecting boneand cartilage formation and regeneration, wound healing,neovascularization and treating FGFR related skeletal and proliferativedisorders.

It is yet another object to provide methods for the use of FGF variantsto prepare medicaments useful in bone and cartilage formation andregeneration, wound healing, neovascularization and treating FGFRrelated skeletal and proliferative disorders.

It is yet a further object of the invention to provide methods for theuse of FGF variants to prepare medicaments useful for targeting to aparticular tissue.

The novel FGF variants provided by this invention fulfill these andother objects.

The present invention is based on the discovery that certainmodifications to members of the FGF family of polypeptides render themadvantageous in that they have enhanced receptor specificity and/oraffinity and altered biological activity.

Unexpectedly, certain FGF variants of the present invention were foundto exhibit enhanced biological activity in addition to receptorselectivity. FGF ligands having enhanced biological activity andincreased receptor selectivity are desired for treatment of variouspathological conditions. Generation of highly active, receptor-specificligands would be useful for the purpose of developing medicaments foruse in tissue repair and regeneration, wound and ulcer healing, bonefracture healing, osteoporosis and other skeletal disorders. Inaddition, the highly active receptor specific ligands are useful for thegrowth, selection, proliferation and differentiation of certain celltypes including chondrocytes, osteoblasts, progenitor cells and stemcells, in vitro and in vivo.

As disclosed in copending international patent application WO 02/36732certain modifications to the polypeptide sequence provide FGF variantswith enhanced receptor specificity which retain biological activity.Specifically, FGF-9 variants comprising mutations in the loop betweenthe β8 and β9 strands of the polypeptide, previously identified as aconserved receptor binding site, and analogous loops in the othermembers of the FGF family, unexpectedly provide enhanced receptorsubtype specificity.

The present invention is related to a variant of FGF having at least oneamino acid substitution in the beta 8-beta 9 loop, said FGF variantcharacterized in at least one of the following attributes compared tothe corresponding wild type FGF: enhanced specificity for one receptorsubtype; increased biological activity mediated by at least one receptorsubtype with equivalent or reduced activity mediated through anotherreceptor subtype; enhanced affinity to at least one receptor subtype;increased cell proliferation mediated through one receptor subtype.

The present invention is directed to novel variants of FGF, and inparticular to variants of FGF-2, FGF-4 and FGF-9. It is now unexpectedlydisclosed that FGF-2 variants comprising at least one mutation in theloop between the β8 and β9 strands, herein defined as the β8-β9 loop,provide superagonist properties in addition to enhanced receptor subtypespecificity. The variants exhibit enhanced receptor subtype specificityfor one receptor subtype compared to the corresponding wild type FGF, byincreasing the biological activity mediated by at least one receptorsubtype while retaining or reducing the activity mediated throughanother receptor subtype. According to one currently preferredembodiment of the present invention the FGF-2 variant comprises an aminoacid (aa) substitution wherein asparagine 111 (Asn111, N111) is replacedwith another amino acid residue thereby providing receptor specificity.These variants are herein denoted FGF2-N111X, having SEQ ID NO:1,wherein X is an amino acid other than asparagine. According to onecurrently preferred embodiment X is arginine (Arg, R) or Glycine (Gly,G).

The abbreviations used herein correspond to the one letter amino acidcode followed by the number designating the amino acid position in the155 aa form of FGF-2 and the one letter amino acid code for thesubstituted amino acid.

A currently preferred embodiment of the present invention provides avariant of FGF-2, denoted herein FGF2-N111R, having SEQ ID NO:2, whereinsubstitution of the asparagine 111 with Arginine (Arg, R) showsessentially unchanged activity towards FGFR3 and FGFR2 while increasingactivity for FGFR1.

A currently more preferred embodiment of the present invention providesa variant of FGF-2, denoted herein FGF2-N111G, having SEQ ID NO:3,wherein substitution of the asparagine 111 with Glycine (Gly, G) showsessentially unchanged activity towards FGFR3 while increasing activityfor FGFR1, and to a lesser extent towards FGFR2.

Another currently more preferred embodiment of the present inventionprovides a variant of FGF-2, denoted herein FGF2(3,5Q)-N111X, having SEQID NO:4, wherein alanine 3 and serine 5 are replaced by glutamine, andasparagine 111 is other asparagine. According to one currently preferredembodiment X is arginine (Arg, R) or Glycine (Gly, G). A currently mostpreferred embodiment of the present invention provides a variant ofFGF-2, FGF2(3,5Q)-N111G, having SEQ ID NO:5, wherein alanine 3 andserine 5 are replaced by glutamine, and asparagine 111 is substitutedwith Glycine (Gly, G) showing essentially unchanged activity towardsFGFR3IIIb and FGFR2 while increasing activity for FGFR1 and FGFR3IIIc.

The FGF-2 variants are shown to stimulate proliferation of chondrocytesand induce differentiation of neuronal cells and may be used tospecifically induce proliferation or differentiation of progenitor cellsand embryonic or adult stem cells.

A comparable amino acid substitution is disclosed for FGF-4. FGF-4, alsoknown as HST and K-FGF, is expressed as a 206 aa precursor proteinhaving a 27 aa signal sequence. An FGF-4 molecule, having 179 aa,comprising at least one mutation in the β8-β9 loop provides a variantwith improved biological activities. According to one currentlypreferred embodiment of the present invention the FGF-4 variantcomprises an amino acid substitution wherein asparagine 165 (Asn165,N165) is replaced with another amino acid residue thereby providingenhanced biological activity. These variants are herein denotedFGF4-N165X, having SEQ ID NO:6, wherein X is an amino acid other than N(asparagine), preferably R (arginine). A currently more preferredembodiment of the present invention provides a 152 aa form of theprotein comprising a 54 amino acid N-terminus truncation in addition tothe N165X substitution. These variants are denoted hereinL55M-FGF4-N165X, having SEQ ID NO:7. Amino acid numbering of the FGF-4variants is according to the 206 aa form.

The L55M-FGF4-N165X variant shows a substantial increase in activitytoward FGFR3 with unchanged activity towards the FGFR1 and a slightreduction in activity towards the FGFR2.

Preferably the variants have at least 2-fold the activity of the nativeFGF-2 in terms of proliferation of FGFR bearing cells induced by thevariant.

The therapeutic utility of these novel FGF-2 and FGF-4 variants isdisclosed for both normal and abnormal FGF receptors, including but notlimited to bone regeneration and bone fracture healing, articularchondrocyte repair, osteoporosis, wound healing, ischemic tissue repair,neural tissue survival and repair and neovascularization.

According to the principles of the present invention it is now disclosedthat through introduction of a single amino acid substitution within theβ8-β9 loop, an FGF polypeptide may undergo interconversion from amitogen to a differentiation factor, or from a differentiation factor toa mitogen. This unexpected property of the novel variants warrants theiradvantageous use in selectively inducing proliferation anddifferentiation of various cell types. The variants of the presentinvention may be used in vitro or in vivo, alone or in combination toachieve a desired effect of proliferation and/or differentiation.Furthermore, the introduction of an amino acid substitution into theβ8-β9 loop of the other members of the FGF family of polypeptides cansimilarly be used to achieve interconversion of a proliferation factorinto a differentiation factor, and a differentiation factor into aproliferation factor.

By way of non-limiting examples, the FGF2-N111X variants, includingFGF2(3,5Q)-N111X, are more potent mitogens than the native FGF2.Alternatively, certain FGF9 variants that were disclosed in PCTapplication WO 02/36732 have now unexpectedly been shown to inducedifferentiation of articular chondrocytes whereas the wild type proteinFGF-9 is both a weak mitogen and a weak differentiation factor. Thesevariants are denoted herein FGF9-W144G, having SEQ ID NO:8 andL37-FGF9-W144X having SEQ ID NO:9. In neuronal cells, the FGF-2 variantof the present invention, FGF2-N111R, is shown to be a more potentdifferentiation factor than FGF-2, as determined by neurite outgrowth.

Currently preferred embodiments in accordance to the inventioncomprising variant forms of FGF-2 and FGF-4 are denoted herein asfollows:

-   -   1) FGF2-N111X (SEQ ID NO:1) having 155 aa wherein Asn (N) at        position 111 is replaced by X, wherein X is an amino acid other        than Asn. The currently preferred amino acid substitution is        selected from X=Gly (G) or Arg (R).    -   2) FGF2-N111G (SEQ ID NO:2) having 155 aa wherein Asn (N) at        position 111 is replaced by Gly (G) or Arg (R).    -   3) FGF2-N111R (SEQ ID NO:3) having 155 aa wherein Asn (N) at        position 111 is replaced by Arg (R).    -   4) FGF2(3,5Q)-N111X (SEQ ID NO:4) having 155 aa wherein Ala3 and        Ser5 are replaced with Glu (Q) and Asn (N) at position 111 is        replaced by X, wherein X is an amino acid other than Asn. The        currently preferred amino acid substitution is selected from        X=Gly (G) or Arg (R).    -   5) FGF2(3,5Q)-N111G (SEQ ID NO:5) having 155 aa wherein Ala3 and        Ser5 are replaced with Glu (Q) and Asn (N) at position 111 is        replaced by Gly (G).    -   6) FGF4-N111X (SEQ ID NO:11) having 179 aa and the Asn (N) at        position 165 is replaced by X. The currently preferred amino        acid substitution is X=Gly (G).    -   7) L55M-FGF4-N111X (SEQ ID NO:12) having 152 aa wherein 54 amino        acids are truncated from the N-terminus, the Leu (L) at position        55 is replaced by a Met (M) and Asn (N) at position 165 is        replaced by X. The currently preferred amino acid substitution        is X=Gly (G).

Additionally, certain variants disclosed in PCT application WO 02/36732are now shown to be effective in selectively inducing proliferation anddifferentiation of cells. The amino acid sequences of the variants aredenoted herein as follows:

(SEQ ID NO: 8) 8) W144X-FGF9 (SEQ ID NO: 9) 9) L37M-W144X-FGF9

The focus of the FGF receptors as receptors involved in certain cancershas raised the unmet need for ligands specific for these receptors;preferably a ligand which binds to one FGFR with high specificity anddoes not substantially bind to the other FGFRs. The high-specificityligand is able to target a receptor on the surface of a specific tissueor organ. The targeting polypeptides are fusion proteins, chimeras,hybrid proteins or conjugates.

Unexpectedly, certain FGF variants of the present invention were foundto retain binding affinity to specific FGF receptors while exhibitingreduced receptor-mediated biological activity, providing variants usefulfor targeting bioactive agents including polypeptides, peptides andanalogs and drugs to specific tissue. Effectively, the variantpolypeptides are useful as carriers which can be used for site-specificdelivery and concentration of bioactive agent to cells, tissues, ororgans in which a therapeutic effect is desired to be effected.

Certain modifications to the FGFs generate polypeptides with improvedproperties including high binding affinity, modified biological activitysuch as reduced stimulation of proliferation and enhanced receptorspecificity.

According to the principles of the present invention it is now disclosedthat mutations in the loop between the β8 and β9 strands of FGFs, hereindefined as β8-β9, previously determined to comprise a major conservedbinding site demonstrated to interact with FGF receptors, and analogousloops in the other members of the FGF family, provide enhanced receptorsubtype specificity and or affinity. According to the principles of thepresent invention it is now disclosed that truncated FGF variantsexhibit reduced activity in promoting growth of receptor bearing cellsthan their corresponding full-length wild type parent growth factor andare particularly useful for targeting bioactive agents to cells, tissuesand organs. Truncated variants of the invention that are most preferredmay further comprise at least one mutation in at least one binding siteto the receptor and are more receptor-selective than the correspondingfull length wild type growth factor. In certain indications, includingsome skeletal and proliferative diseases, it is advantageous to useinactive ligands for targeting in order to avoid activation of receptorswhere activation of said receptors may advance the diseased state.According to one aspect of the present invention said FGF variantswherein the N- and/or C-termini are truncated such that the truncationextends near to or within the core domain provide molecules with reducedbiological activity useful as an antagonist of FGFR or for targetingbioactive agents to specific cells or tissues or organs. An FGF-9variant having a 63 amino acid N-terminus truncation, is denoted hereinR64M-FGF9, having SEQ ID NO:10. The R64M-FGF9 variant was disclosed incopending PCT patent application WO 02/36732 as the shortest variant ofFGF-9 having biological activity and improved binding specificity towardFGFR3. The present invention relates to additional beneficial propertiesof the R64M-FGF9 variant, specifically for use as a targeting moleculespecific for FGFR3.

A currently more preferred variant of the present invention, having a 63amino acid truncation and an 18 amino acid C-terminus truncation isdenoted herein FGF9-2, having SEQ ID NO:11. The FGF9-2 variant wasdisclosed in copending PCT patent application WO 02/36732 as a variantof FGF-9 having reduced biological activity. The present inventiondiscloses unexpected additional beneficial properties of the FGF9-2variant, specifically for use as a targeting molecule specific forFGFR3.

According to yet another currently preferred embodiment of the inventionthere is provided an FGF comprising a substitution of at least oneresidue in a major binding site of the molecule to the receptor inconjunction with a truncation of the N- and/or C-termini. An amino acidsubstitution according to the invention affects binding of the variantto one receptor but not to another thereby providing a basis forreceptor specific mutants of FGFs.

The preferred FGF variant has enhanced specificity for one receptorsubtype compared to the corresponding wild type parent FGF, bydecreasing the biological activity mediated by at least one receptorsubtype while retaining the activity mediated through another receptorsubtype. The truncated molecule exhibits reduced biological activitywhile maintaining high receptor affinity.

In a non-limiting example it is possible to diminish the biologicalactivity resulting from FGF-9 binding to FGFR1 while retaining bindingto FGFR3. Preferably the binding to FGFR3 is a high affinity bindingwith reduced biological activity. More preferably the binding to FGFR3is a high affinity binding with no biological activity.

Preferably the mutation results in a substitution of tryptophan 144(W144) of the β8-β9 loop as numbered according to wild type parentFGF-9, or an amino acid in the corresponding position of the β8-β9 loopof an FGF. More preferably the mutation is in the β8-β9 loop of FGF-2,FGF-4 or FGF-9. Here we disclose increased receptor specificity by apoint mutation in FGF-9 resulting in an amino acid substitution in theloop between the β8 and β9 strands. The variants are furthermoretruncated at the N- or C-terminus or both termini wherein the biologicalactivity is reduced but the affinity to the receptor is substantiallyunaffected.

According to one currently most preferred embodiment of the presentinvention FGF-9 comprises an amino acid substitution wherein Trp144(W144) is replaced with other amino acid residues providing receptorspecificity and N-terminal and/or C-terminal truncation(s) that reducethe biological activity and retain receptor affinity. Introduction ofglycine at position 144 of FGF-9 abolishes its binding to FGFR1, whileretaining significant affinity towards FGFR3 and to a lesser extent,FGFR2. According to an additional currently preferred embodiment theR64M-FGF9 variant further comprises a W144 substitution. This variant isdenoted herein R64M-FGF9-W144X, having SEQ ID NO:10, wherein Trp144 issubstituted with amino acid residues including, but not limited toglycine (G), arginine (R), valine (V) or glutamate (E) that abolish thebinding to FGFR1 while retaining high affinity binding to FGFR3 and alesser affinity to FGFR2. These variants, having reduced biologicalactivity and high receptor affinity are denoted herein R64M-FGF9-W144G,R64M-FGF9-W144R, R64M-FGF9-W144V and R64M-FGF9-W144E.

According to an additional currently more preferred embodiment of thepresent invention the FGF9-2 variant further comprises a W144substitution. This variant is denoted herein FGF9-2-W144X, having SEQ IDNO: 12. The FGF9-2 variant further comprises an amino acid substitutionwherein Trp144, or the equivalent position in other FGFs, is substitutedwith amino acid residues including, but not limited to glycine (G),arginine (R), valine (V) or glutamate (E) that abolish the binding toFGFR1 while retaining high affinity binding to FGFR3 and a lesseraffinity to FGFR2. These variants, having reduced biological activityand high receptor affinity are denoted herein FGF9-2-W144G,FGF9-2-W144R, FGF9-2-W144V and FGF9-2-W144E.

Another aspect of the invention provides a substitution of anotherresidue in the β8-β9 loop, namely the amino acid adjacent to Trp144,asparagine 143 (Asn143 or N143) of FGF-9, or the equivalent position inother FGFs, with another amino acid residue including, but not limitedto serine, to diminish binding to FGFR1 while retaining high affinitybinding to FGFR3 and a lesser affinity to FGFR2. Furthermore,truncations reduce biological activity and retain binding capacity ofthe FGF. These variants are denoted herein R64M-FGF9-N143X, andF9-2-N143X, having SEQ ID NOS:14 and 15, respectively, wherein X isother than asparagine (N) and more preferably serine (S).

Further preferred variants comprise analogous polypeptides of FGF-2, inparticular variants comprising the 120 aa core domain and truncations atboth the N- and C-termini.

A currently preferred embodiment of the present invention provides acomposition useful to target bioactive agents to particular cells,tissues and organs. A currently more preferred embodiment comprises acovalent conjugate or chimeric recombinant (fusion protein) comprisingan FGF variant linked to a bioactive agent. This link may be via adirect bond, including a peptide bond, and the bioactive agent may be adetectable label, cytotoxic drug, a pharmaceutically active compound ordiagnostic compound. These include, but are not limited to, peptides andpeptide analogs, peptidomimetics, oligopeptides, proteins, apoproteins,glycoproteins, antigens and antibodies or antibody fragments, receptorsand other membrane proteins, aptamers, enzymes, coenzymes, enzymeinhibitors including tyrosine kinase inhibitors, amino acids and theirderivatives, hormones, lipids, phospholipids, toxins and anti-cancerdrugs.

In a currently most preferred embodiment the composition compriseschimera or conjugate of R64M-FGF9-W144G or FGF9-2-W144G linked to apeptide or peptide analog.

A currently preferred embodiment of the present invention is acomposition useful to increase the size of a bone growth plate inabnormal bone. A currently more preferred embodiment of the presentinvention is a pharmaceutical composition comprising as an activeingredient a covalent conjugate or chimeric recombinant comprising anFGF variant linked to a bioactive agent. This link may be via a directbond, including a peptide bond, and the bioactive agent may be adetectable label, cytotoxic drug, a pharmaceutically active compound ordiagnostic compound. These include, but are not limited to thosebioactive agents previously listed. In one currently most preferredembodiment the abnormal bone is a dysplasic bone, the FGF variant isselected from R64M-FGF9 or FGF9-2, and the bioactive agent is anatriuretic peptide. In another currently most preferred embodiment theFGF variant is selected from R64M-FGF9-W144G or FGF9-2-W144G and thebioactive agent is selected from C-type natriuretic peptide (CNP) or ananalog thereof.

The present invention further provides variants useful for regulatingactivity of a mutated FGF Receptor. A currently preferred embodiment ofthe present invention is the use of a variant of the invention that actsas an antagonist to reduce the activity of a mutated receptor indiseases and disorders related to FGFR. A currently more preferredembodiment of the present invention is the use of a variant of theinvention that acts as an antagonist to reduce the activity of a mutatedFGFR3 in diseases and disorders related to FGFR3, includingachondroplasia, thanatophoric dysplasia and proliferative diseasesincluding transitional cell carcinoma (TCC) of the bladder, breastcancer and multiple myeloma.

Currently most preferred embodiments in accordance to the inventioncomprising variant forms of FGF are denoted herein as follows:

-   -   10) R64M-FGF9 (SEQ ID NO:10). The sequence of this variant has        been disclosed in PCT application WO 02/36732.    -   11) R64M-FGF9-W144X (SEQ ID NO:11) having 145aa with a        truncation of 63 amino acids from the N-terminus, the Arg (R) at        position 64 of the wild type parent FGF-9 replaced by Met (M)        and wherein X at position 144 of the wild type parent FGF-9 is        other than Trp (W) and more preferably selected from Glycine        (G), Arg (R), Val (V) or Glu (E).    -   12) FGF9-2 (SEQ ID NO:12). The sequence of this variant has been        disclosed in copending PCT application WO 02/36732.    -   13) FGF9-2-W144X (SEQ ID NO:13) having 127aa with a truncation        of 63 amino acids from the N-terminus, the Arg (R) at position        64 of the wild type parent FGF-9 replaced by Met (M) and a        truncation of 18 amino acids from the C-terminus, the Pro (P) at        position 191 of the wild type parent FGF-9 replaced with a        termination signal and wherein X at position 144 of FGF-9 is        other than Trp (W), wherein the currently preferred amino acid        substitution is selected from Gly (G), Arg (R), Val (V) or Glu        (E).    -   14) R64M-FGF9-N143X (SEQ ID NO:14) having 145aa with a        truncation of 63 amino acids from the N-terminus, the Arg (R) at        position 64 of the wild type parent FGF-9 replaced by Met (M)        and wherein X at position 143 of the wild type parent FGF-9 is        other than Asn (N) and more preferably Ser (S).    -   15) FGF9-2-N143X (SEQ ID NO:15) having 127aa with a truncation        of 63 amino acids from the N-terminus, the Arg (R) at position        64 of the wild type parent FGF-9 replaced by Met (M), a        truncation of 18 amino acids from the C-terminus, the Pro (P) at        position 191 of the wild type parent FGF-9 replaced with a        termination signal and wherein X at position 143 of FGF-9 is        other than Asn (N), and more preferably Ser (S).

A currently preferred embodiment of the present invention is a method totarget bioactive agents to particular cells, tissues and organs. In acurrently more preferred embodiment a composition comprising an FGFcomplex molecule comprising a covalent conjugate or chimeric recombinantcomprising an FGF variant linked to a bioactive agent is administered toa patient in need thereof. In a currently most preferred embodiment thecomposition comprises an R64M-FGF9-W144G or FGF9-2-W144G and peptide orpeptide analog chimera or conjugate.

A currently preferred embodiment of the present invention is a method toincrease the size of a bone growth plate in abnormal bone by treatingthe bone with a pharmaceutical composition comprising a covalentconjugate or chimeric recombinant FGF complex molecule comprising an FGFvariant linked to a bioactive agent, further comprising apharmaceutically acceptable diluent, carrier and/or stabilizer. In acurrently more preferred embodiment the abnormal bone is a dysplasicbone, the FGF variant is R64M-FGF9-W144G or FGF9-2-W144G and thebioactive agent is C-type natriuretic peptide (CNP) or an analogthereof. According to one aspect of the invention, the FGF variant is 5′to the CNP, in another aspect the FGF variant is 3′ to the CNP. It is tobe understood that the CNP analogs include the CNP(1-22) 22 aa peptide,an active CNP(5-22) 17 aa peptide or an active variant thereof.

The amino acid sequences of currently preferred FGF complex molecules,followed by the polynucleotide sequences, are presented herein asfollows:

-   -   16) FGF9-2-W144X-CNP(1-22) (SEQ ID NO:16) having 152 aa        comprising SEQ ID NO:16, linked to a 22 aa CNP molecule or a        stable derivative thereof via a polypeptide linker.    -   17) CNP(1-22)-FGF9-2-W144X (SEQ ID NO:17) having at least 157 aa        comprising a CNP(1-22) molecule or a stable derivative thereof,        linked to SEQ ID NO:16 via a polypeptide linker.

It is to be understood that a complex molecule comprises an FGF varianthaving SEQ ID NOS:12-15 linked to a bioactive agent as either theN-terminal component or the C-terminal component of the covalentconjugate or chimeric recombinant. It is further understood that alinker may be a polypeptide linker such as those known in the art. Acurrently preferred embodiment comprises an FGF variant having SEQ IDNOS:12-15 linked to a bioactive agent as either the N-terminal componentor the C-terminal component via a polyglycine linker of 2-20 aminoacids.

The polynucleotide sequences corresponding to the novel variants isdisclosed herein as follows:

(SEQ ID NO: 18) 18) FGF2-N111X DNA (SEQ ID NO: 19) 19) FGF2-N111G DNA(SEQ ID NO: 20) 20) FGF2-N111R DNA (SEQ ID NO: 21)21) FGF2(3, 5Q)-N111X DNA. (SEQ ID NO: 22 22) FGF2(3, 5Q)-N111G DNA).(SEQ ID NO: 23) 23) FGF4-N111X DNA (SEQ ID NO: 24)24) L55M-FGF4-N165X DNA. (SEQ ID NO: 25)25) R64M-FGF9 DNA disclosed in PCT application WO 02/36732.(SEQ ID NO: 26) 26) R64M-FGF9-W144X DNA (SEQ ID NO: 27)27) FGF9-2 DNA disclosed in PCT application WO 02/36732. (SEQ ID NO: 28)28) FGF9-2-W144X DNA. (SEQ ID NO: 29) 29) R64M-FGF9-N143X DNA.(SEQ ID NO: 30) 30) FGF9-2-N143X DNA. (SEQ ID NO: 31)31) FGF9-2-W144X-CNP(1-22) DNA (SEQ ID NO: 32)32) CNP(1-22)-FGF9-2-W144X DNA

According to one currently preferred embodiment of the present inventiona pharmaceutical composition comprising as an active ingredient at leastone variant having SEQ ID NOS:1-17 and a pharmaceutically acceptablecarrier, diluent or excipient is provided.

In a currently more preferred embodiment of the present invention thevariants having SEQ ID NOS:1-7 are formulated to provide pharmaceuticalcompositions useful for promoting or accelerating repair or regenerationof endochondral bone, intramembranous bone, articular cartilage, spinaldefects and other skeletal disorders and for promoting or acceleratingneovascularization in indications including burns, cuts, lacerations,bed sores, ulcers such as those seen in diabetic patients, repair andregeneration of tissue, including skeletal, nerve and vascular tissue.According to yet a further aspect of the present invention is a methodof promoting or accelerating bone growth or cartilage repair whichcomprises administering to a patient a therapeutically effective amountof a pharmaceutical composition in combination with a matrix device. Thematrix may be synthetic or natural. In a non-limiting example the matrixis a plasma protein matrix or a calcium phosphate matrix. Thus thepresent method may be used to promote tissue regeneration and repairincluding cartilage, bone and wound healing.

According to another aspect of the present invention it is disclosedthat the pharmaceutical compositions comprising at least one variant FGFof the present invention having SEQ ID NOS:10-17 have improvedtherapeutic utility in diseases and disorders associated with FGFreceptors. The therapeutic utility of these novel variants is disclosedin diseases involving both normal and abnormal FGF receptors, includingbut not limited to skeletal disorders including but not limited toAchondroplasia, Hypochondroplasia, and osteoporosis and proliferativediseases and disorders.

According to yet another aspect of the present invention it is disclosedthe preferred variants having SEQ ID NO:1-9 have improved utility in theselective induction of proliferation and differentiation of cells. Theuse of these variants is disclosed for in vitro or in vivo treatment ofcells.

According to yet a further aspect of the present invention is a methodof promoting or accelerating neovasculogenesis which comprisesadministering to a patient a therapeutically effective amount of apharmaceutical composition comprising a variant of the present inventionand optionally a matrix-free or matrix device. The matrix may besynthetic or natural. In a non-limiting example the matrix is a plasmaprotein matrix or a calcium phosphate matrix. Thus the present methodmay be used to promote tissue regeneration and repair includingcartilage, bone and wound healing.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the proliferative activity of FGF2-N111R.

FIGS. 2A and 2B show the mitogenic activity induced by the FGF-2variant, FGF2(3,5Q)-N111G on FGFR-transfected FDCP cells.

FIG. 3 depicts the mitogenic activity of the FGF-2 variantFGF2(3,5Q)-N111G on FGFR-transfected FDCP cells as a function of heparinconcentration.

FIG. 4 shows the mitogenic activity induced by the FGF-4 variant,FGF4-L55M-N165R on FGFR-transfected FDCP cells.

FIG. 5A displays the electrophoresis pattern of FGF-9 variants onSDS-PAGE.

FIG. 5B shows the reduced mitogen activity of the truncated FGF-9variants.

FIG. 6 shows the results of a competition binding assay of FGF-9variants.

FIGS. 7A and 7B show two exposures of the distribution of I¹²⁵FGF9-2-W144G variant in the mouse growth plate following IP delivery.FIG. 7A shows the signal distribution while FIG. 7B shows the outline ofthe cells.

FIG. 8 depicts a proliferation curve of human articular chondrocytesgrown in the presence of the variants of the present invention.

FIGS. 9A-9E show the phenotype of human articular chondrocytes grown inthe presence of variants of the present invention.

FIGS. 10A-10C show the phenotype of porcine articular chondrocytes grownin the presence of variants of the present invention.

FIGS. 11A-11C show the phenotype of porcine articular chondrocytes grownin the presence of variants of the present invention, phalloidinstaining.

FIGS. 12A-12C show the expression of collagen type II protein in aporcine chondrocyte pellet culture.

FIGS. 13A-13D show proteoglycan expression in porcine articularchondrocyte pellet culture, as determined by toluidine blue staining.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Fibroblast growth factors (FGFs) constitute a large family ofstructurally related, heparin binding polypeptides, which are expressedin a wide variety of cells and tissues. Overall, the FGFs share between17-72% amino acid sequence homology and a high level of structuralsimilarity. A homology core of around 120 amino acids is highlyconserved and has been identified in all members of the family. Theresidues of the core domain interact with both the FGFR and heparin.Twelve antiparallel β strands have been identified in the corestructure, labeled β1 through β12, linked one to another by loops ofvariable lengths, organized into a trefoil internal symmetry. Sequencealignment and location and length of the β strands for FGF-1 throughFGF-19 is depicted in FIG. 6 of Plotnikov et al. (Cell 101, 413, 2000).

According to the principles of the present invention it is now disclosedthat FGF variants of the present invention comprising amino acidsubstitutions in the loop between the β8 and β9 strands of the corestructure yield variants with improved properties, in addition toaltered specificity to FGFRs. In certain embodiments the amino acidsubstitution yields active variants with superagonist properties. Thevariants thus obtained will have improved properties in terms ofreceptor specificity, stability or affinity in addition to enhancedmitogenic activity or differentiation potential. Furthermore, thevariants so obtained may further comprise additional modificationswithin or outside of the β8-β9 loop providing variants with improvedstability, solubility or yield.

The FGF ligands with enhanced biological activity and increased receptorselectivity are highly necessary for treatment of various pathologicalconditions. The variants would be useful for the purpose of research aswell as for the purpose of developing possible medicaments for use intissue repair and regeneration, wound and ulcer healing, bone andcartilage disorders, bone fracture healing, osteoporosis and otherskeletal disorders.

Further disclosed are FGF variants which retain binding affinity tospecific FGF receptors without stimulating receptor-mediated biologicalactivity, providing FGF variants useful as receptor antagonists or fortargeting bioactive agents including polypeptides, peptides and analogsand drugs to specific tissue. The variants with reduced activity areuseful as antagonizing a specific receptor in indications related toabnormal FGFR activation. Moreover, the variant polypeptides are usefulas carriers which can be used for site-specific delivery andconcentration of bioactive agent to cells, tissues, or organs in which atherapeutic effect is desired to be effected. Certain modificationsyield polypeptides with improved properties including high bindingaffinity, reduced biological activity and enhanced receptor specificity,thus providing therapeutically beneficial molecules for treatingskeletal disorders, including but not limited to achondroplasia, andproliferative diseases including but not limited to multiple myeloma,transitional cell carcinoma (TCC) of the bladder, breast cancer andcervical carcinoma. The targeting polypeptides are fusion proteins,chimeric recombinants, hybrid proteins or conjugates. For conveniencecertain terms employed in the specification, examples and claims aredescribed here.

As used herein and in the claims the term “FGFR” denotes a receptorspecific for FGF which is necessary for transducing the signal exertedby FGF to the cell interior, typically comprising an extracellularligand-binding domain, a single transmembrane helix, and a cytoplasmicdomain that contains a tyrosine kinase activity. The term “FGFR”includes soluble versions comprising the extracellular domain andlacking the transmembrane and kinase domains, and other variantsthereof.

As used herein and in the claims the term “inactive FGF” denotes an FGFmolecule or variant which after binding to an FGF receptor elicitsstimulation of mitogenesis at most half that of the same cells exposedto the wild type parent FGF molecule, as measured in cell based assaysknown in the art. More preferably, the variant elicits stimulation ofmitogenesis at most one quarter that of the same cells exposed to thewild type parent FGF molecule.

As used herein and in the claims the term “FGF receptor specificity”denotes the fact that a certain FGF molecule binds to a particular FGFreceptor and elicits a receptor mediated biological response at aconcentration at least twice as high as its activity upon binding toanother FGFR. Biological responses are measured by methods known in theart.

The term “affinity” as used herein denotes the ability of a ligand orvariant of said ligand to bind to a specific receptor. Modifications toa ligand which stabilize favorable conformation or enhance amino acidside chain interactions will result in increased receptor affinity whilethose which destabilize favorable conformation or decrease amino acidside chain interactions will result in decreased receptor affinity. Acompetitive binding assay was established to determine the relativeaffinity of FGF variants compared to that of wild type parent FGFtowards an FGF receptor. Variants having high affinity for an FGFreceptor and reduced mitogenic activity are designated potential FGFantagonists.

As used herein the term “differentiation factor” refers to a substance,in particular a polypeptide, which determines the fate that a cell willacquire upon exposure to that substance, alone or in combination withother substances. In a non-limiting example, differentiation isdetermined by morphological and phenotypic changes or by biochemical ormolecular changes.

As used herein the term “mitogen” or “proliferation factor” refers to asubstance that induces an increase in the number of cells.

As used herein and in the claims the term “core”, “core domain” or “corestructure” denotes a region of homology of around 120 amino acids thatis found in all native FGFs. Twenty eight amino acid residues are highlyconserved and six are identical. Twelve structurally conservedanti-parallel β strands have been identified in all the FGFs. The coredomain comprises the FGFR and heparin binding sites.

As used herein and in the claims the term “beta8-beta9” or “β8-β9” or“β8-β9 loop” refers to the loop of 2 to 5 amino acid residues that liebetween the eighth and ninth β-pleated strands of the core structure asdisclosed herein.

As used herein and in the claims the terms “amino terminus” and“N-terminus” of a polypeptide may be used interchangeably. Similarly,the terms “carboxy terminus” and “C-terminus” may be usedinterchangeably.

“Nucleic acid sequence” or “polynucleotide” as used herein refers to anoligonucleotide or nucleotide and fragments or portions thereof, and toDNA or RNA of genomic or synthetic origin which may be single- ordouble-stranded, and represent the sense or antisense strand. Similarly,“amino acid sequence” as used herein refers to an oligopeptide, peptide,polypeptide, or protein sequence, and fragments or portions thereof, andto naturally occurring, synthetic or recombinant molecules. The termslisted herein are not meant to limit the amino acid sequence to thecomplete, wild type amino acid sequence associated with the recitedprotein molecule. The term “variant” as used herein refers to apolypeptide sequence that possesses some modified structural property ofthe wild type or parent protein. For example, the variant may betruncated at either the amino or carboxy terminus- or both termini ormay have amino acids deleted, inserted or substituted. It may beantagonistic or agonistic with respect to normal properties of thenative protein. An antagonist is defined as a substance that binds tobut does not activate a receptor mediated response. An agonist isdefined as a substance induces a receptor-mediated response similar tothat induced by the wild type ligand. A superagonist is defined as asubstance that induces a cellular or physiological response at aconcentration at least half that observed with the wild type protein.More preferably, a cellular or physiological response is elicited at aconcentration at least four fold less than that observed with the wildtype protein. A biological response may be, for example, the stimulationof cell division, differentiation, angiogenesis or wound repair. Abiological response may encompass other functional properties of thewild type parent protein and would be well known to those practicing theart.

It is contemplated in this invention that a variant may have alteredbinding to a receptor compared to that of the wild type parent protein.This binding may enhance or depress a biological response. Accordingly,the variant may have altered specificity for one or more receptors.

The variant may be generated through recombinant DNA technologies, wellknown to those skilled in the art. As used herein, the term “polymerasechain reaction” (“PCR”) refers to the methods disclosed in U.S. Pat.Nos. 4,683,195; 4,683,202 and 4,965,188 hereby incorporated byreference.

The term “expression vector” and “recombinant expression vector” as usedherein refers to a recombinant DNA molecule containing a desired codingsequence and appropriate nucleic acid sequences necessary for theexpression of the operably linked coding sequence in a particular hostorganism. The expression vector may comprise sequences encodingheterologous domains including but not limited to protein detection,purification or cleavage sequences that may be fused at the N- orC-terminus to the desired coding sequence, to yield a fusion protein. Itis contemplated that the present invention encompasses expressionvectors that are integrated into host cell genomes, as well as vectorsthat remain unintegrated into the host genome.

As used herein, the “amino acids” used in the invention are those whichare available commercially or are available by routine syntheticmethods. Certain amino acid residues may require special methods forincorporation into the peptide, and sequential, divergent or convergentsynthetic approaches to the peptide sequence are useful in thisinvention. Natural coded amino acids and their derivatives arerepresented by either the one-letter code or three-letter codesaccording to IUPAC conventions. When there is no indication, the Lisomer was used. Other pharmaceutically active amino acids, includingsynthetic amino acids, are known in the art and are intended to beincluded in the invention.

As used herein and in the claims a “bioactive agent” is any agent whichis desired to be delivered to cells, tissues or organs for modulating ormodifying cell function, including for therapeutic effects. Inaccordance with the present invention, bioactive agents include, but arenot limited to, pharmaceutically active compounds or diagnosticcompounds. These include, but are not limited to, peptides and peptideanalogs, peptidomimetics, oligopeptides, proteins, apoproteins,glycoproteins, antigens and antibodies or antibody fragments thereto,receptors and other membrane proteins, aptamers, enzymes, coenzymes,enzyme inhibitors, amino acids and their derivatives, hormones, lipids,phospholipids, liposomes; toxins such as ricin or ricin fragments,aflatoxin, digoxin, xanthotoxin, rubratoxin, ribosome inactivatingproteins; tyrosine kinase inhibitors, photoreactive agents, antibioticssuch as cephalosporins, penicillin and erythromycin; analgesics andanti-inflammatory substances; antimicrobial agents; antihypertensiveagents; antiviral agents; antihistamines; anti-cancer drugs includingchemotherapeutic agents, such as chlorambucil, carboplatin, derivativesof busulfan, doxorubicin, etoposide, genestein, topotecan (TPT);tranquilizers; neuroprotective agents; antispasmodics; anti-Parkinsonagents; vitamins. Other bioactive agents include nucleotides;oligonucleotides; polynucleotides; and their art-recognized andbiologically functional analogs and derivatives; plasmids, cosmids,artificial chromosomes, other nucleic acid vectors; antisensepolynucleotides including those substantially complementary to at leastone endogenous nucleic acid or those having sequences with a senseopposed to at least portions of selected viral or retroviral genomes;promoters; enhancers; inhibitors; other ligands for regulating genetranscription and translation.

As herein, the terms “bone defect” or “bone disorder” is meant animbalance in the ratio of bone formation to bone resorption, such that,if unmodified, the subject will exhibit less bone than desirable, or thesubject's bones will be less intact than desired. Bone deficit may alsoresult from mutation, fracture, from surgical intervention or fromdental or periodontal disease. By “cartilage defect” or “cartilagedisorder” is meant damaged cartilage, less cartilage than desired, orcartilage that is less intact and coherent than desired. Contemplatedare indications including rheumatoid arthritis, osteoarthritis and kneeinjuries.

Representative uses of the compounds of the present invention include:repair of bone defects and deficiencies, such as those occurring inclosed, open and non-union fractures; prophylactic use in closed andopen fracture reduction; promotion of bone healing in plastic surgery;stimulation of bone ingrowth into non-cemented prosthetic joints anddental implants; elevation of peak bone mass in pre-menopausal women;treatment of growth deficiencies; treatment of peridontal disease anddefects, and other tooth repair processes; increase in bone formationduring distraction osteogenesis; treatment of articular chondrocytesprior to heterologous or autologous transplantation and treatment ofother skeletal disorders, such as age-related osteoporosis,post-menopausal osteoporosis, glucocorticoid-induced osteoporosis ordisuse osteoporosis and arthritis. The compounds of the presentinvention are useful in repair of congenital, trauma-induced or surgicalresection of bone (for instance, for cancer treatment), and in cosmeticsurgery. Further, the compounds of the present invention can be used forlimiting or treating cartilage defects or disorders. Treatment includesdirect application of the variants to the traumatized area or systemictherapy as well as treatment of cells ex vivo and in vitro for tissueengineering and tissue regeneration.

As used herein, the terms “fusion protein” or “chimera”, “chimericrecombinant” or “hybrid” refer to a single polypeptide produced usinghost cells expressing a single polynucleotide encoding an FGF variant ofthe invention and a bioactive agent including a polypeptide, peptide orpeptide analog contiguous and in open reading frame. Certain peptidelinkers may be incorporated to separate the FGF and the bioactivepolypeptide, peptide or peptide analog. Using current methods of geneticmanipulation, a variety of peptides or peptide hormones, includingnatriuretic peptides such as CNP or growth hormone, can be translated asfusion proteins with FGF variants which in turn can specifically targetcells and facilitate internalization. The present invention provides ahighly effective system for delivery of an activity-inducing moiety intoa particular type or class of cells. The fusion proteins generated canbe screened for the desired specificity and activity utilizing themethods set forth in the examples and by various routine procedures. TheFGF variant fusion proteins encoded by the nucleic acids of the presentinvention must be able to specifically bind the selected target cell andinternalize the FGF fusion.

As used herein, the term “conjugate” refers to a bioactive agentcovalently bound to a carrier or targeting moiety. Certain variants ofthe invention provide carriers or targeting agents for bioactive agents.

An FGF “targeting molecule” or “complex molecule” refers to an FGFvariant of the invention linked to a bioactive agent as a recombinantchimera or covalent conjugate.

Provided in the present invention are pharmaceutical compositionscomprising an FGF variant and a bioactive agent as a fusion protein oralternatively an FGF variant conjugate comprising an FGF variant and abioactive agent that are covalently bound useful for FGF targeting. Thepresent invention further provides methods for inhibiting proliferationof cells that express FGFRs comprising administering an FGF varianttargeting composition to the cells. For example the composition isadministered in a therapeutically effective amount to a subject that hasa tumor, wherein the tumor cells express specific FGFR.

FGF activity is conveniently determined using biological assaysperformed in-vitro, ex-vivo and in vivo. The assays are used todemonstrate the activity elicited upon binding of an FGF molecule to itsreceptors. The biological assays routinely used to test activities ofvariant FGFs include, but are not limited to, the following:

-   -   i. binding of variant FGFs to cloned FGF receptors expressed on        immortalized cell lines, thereby eliciting a biological response        including cell proliferation or inhibition of cell        proliferation;    -   ii. cell culture systems;    -   iii. stimulation of bone growth in animal models of bone growth        and cell cultures;    -   iv. enhancement of cartilage repair in animal models of        cartilage disease and trauma.

Design of Variants

One currently preferred embodiment of the invention is an FGF moleculein which an amino acid substitution is incorporated into the β8-β9 loop.Structural data has recently identified that domain as a major bindingsite demonstrated to interact with the link connecting the Ig-like 2(D2) and Ig-like 3 (D3) domains of the receptor (Plotnikov et al., Cell98, 641 1999). Plotnikov et al., (Cell 101, 413, 2000) have shown thatcertain domains in the FGFR such as βC-βE (D2-D3 linker) and βF-βG (D3)regulate FGF-2 binding specificity by interacting with the β4-β5 loopand the amino terminus of FGF. Additionally, FGFR2 makes hydrophobiccontacts with Asn102 of FGF-2 (numbering of aa is according to FGF-2lacking the 9 aa signal peptide; equivalent to N111 as denoted herein)and forms hydrogen bonds with Asn104 (equivalent to N113). An Asn104(N113) substitution led to a 400-fold reduction in binding affinity ofFGF2 for FGFR1.

Surprisingly, the inventors herein disclose an increase in FGFR1activity for a variant having a neighboring N111 substitution in FGF-2.

Table 1 depicts the amino acid alignment of the residues in the β8-β9loop of the known FGFs and 1-3 adjacent residues from the 0 strand oneither side. The Asn111 of FGF-2 AND FGF-4 and the Trp144 of FGF-9 arehighlighted in bold and underlined.

TABLE 1 Amino acid sequence alignment of the β8-β9 and adiacent residues FGF-1LEENHYNTY Residues: 104-112 (SEQ ID NO: 33) FGF-2 LESN N YNTYResidues: 107-115 (SEQ ID NO: 34) FGF-3 IHELGYNTY Residues: 121-129(SEQ ID NO: 35) FGF-4 LLPN N YNAY Residues: 161-169 (SEQ ID NO: 36)FGF-5 FQENSYNTY Residues: 166-174 (SEQ ID NO: 37) FGF-6 LLPNNYNTYResidues: 163-171 (SEQ ID NO: 38) FGF-7 ILENHYNTY Residues: 143-151(SEQ ID NO: 39) FGF-8 VLENNYTAL Residues: 151-159 (SEQ ID NO: 40) FGF-9FEEN W YNTY Residues: 140-148 (SEQ ID NO: 41) FGF-10 IEENGYNTYResidues: 156-164 (SEQ ID NO: 42) FGF-11 VFENYYVLY Residues: 149-157(SEQ ID NO: 43) FGF-12 VFENYYVIY Residues: 151-159 (SEQ ID NO: 44)FGF-13 VFENYYVLY Residues: 147-155 (SEQ ID NO: 45) FGF-14 VFENYYVIYResidues: 149-157 (SEQ ID NO: 46) FGF-15 MDCLGYNQY Residues: 133-141(SEQ ID NO: 47) FGF-16 FEENWYNTY Residues: 139-147 (SEQ ID NO: 48)FGF-17 VLENNYTAF Residues: 133-141 (SEQ ID NO: 49) FGF-18 VLENNYTALResidues: 133-141 (SEQ ID NO: 50) FGF-19 IRPDGYNVY Residues: 126-134(SEQ ID NO: 51) FGF-20 FEENWYNTY Residues: 143-151 (SEQ ID NO: 52)FGF-21 LLEDGYNVY Residues: 127-135 (SEQ ID NO: 53) FGF-22 IEENGHNTYResidues: 119-127 (SEQ ID NO: 54) FGF-23 TLENGYDVY Residues: 119-127(SEQ ID NO: 55) FGF-CX FEENWYNTY Residues: 143-151 (SEQ ID NO: 56) JaffaLLEDGYNVY Residues: 127-135 (SEQ ID NO: 57) Note: The aa numbering ofFGF-2 is according to the 155 aa isoform; amino acid 107 would be 98 inthe 146 aa isoform. Sequence alignment for FGF-1-FGF-19 is according toPlotnikov et al. (Cell 101, 413, 2000). FGF-20-23 sequences wereidentified in Kirikoshi et al. (BBRC 274, 337, 2000), Nishimura et al.(BBA 1492, 203, 2000), Nakatake et al. (BBA 1517, 460, 2001) andYamashita et al. (BBRC 277, 494, 2000), respectively. The FGF-CXsequence is disclosed in WO 01/07595. FGF-18 is also known as zFGF-5.The human FGF Jaffa sequence is disclosed in WO 01/38357.

Preferred Embodiments

As disclosed in copending PCT patent application WO 02/36732, certainmodifications to the polypeptide sequence provide variants with enhancedreceptor specificity which retain biological activity. Specifically, FGFvariants comprising mutations in the loop between the β8 and β9 strands,herein defined as β8-β9, previously determined to comprise a majorbinding site demonstrated to interact with the receptor, and analogousloops in the other members of the FGF family, provide enhanced receptorsubtype specificity. Here we disclose increased receptor specificityand/or affinity and enhanced biological activity of FGF ligands by aminoacid substitutions in the β8-β9 loop, specifically at position 111 ofwild type FGF-2.

Substitution of aligned residues in FGF-2, exemplified by replacing Asn102 (N111 of the 155 aa isoform) with Ala (N102A) (Zhu et al., ProteinEng, 10, 417, 1997) was reported to exhibit no receptor specificityalterations. Disclosed herein are FGF-2 variants wherein the identicalasparagine at position 111 (N111) is substituted with another residueunexpectedly exhibiting both an increase in biological activity andincreased receptor specificity.

A currently preferred embodiment of the invention is denoted FGF2-N111Xwherein X is other than asparagine and more preferably selected fromglycine (Gly, G) or arginine (Arg, R). This sequence of this variant isdenoted herein SEQ ID NO:1. A currently preferred embodiment of thepresent invention provides a variant of FGF-2, denoted herein FGF2-N111Rhaving SEQ ID NO:2, wherein substitution of the asparagine 111 witharginine (Arg, R) shows essentially unchanged activity towards FGFR3 andFGFR2 while increasing activity for FGFR1.

A currently more preferred embodiment of the present invention providesa variant of FGF-2, denoted herein FGF2-N111G having SEQ ID NO:3,wherein substitution of the asparagine 111 with glycine (Gly, G) showsessentially unchanged activity towards FGFR3 while increasing activityfor FGFR1, and to a lesser extent towards FGFR2.

The number designations correspond to the three letter or one-letteramino acid codes followed by the amino acid position in the 155 aminoacid form of FGF-2.

The variants of the invention that are most preferred may furthercomprise additional modifications within, or outside of, the β8-β9 loop.Examples of modifications include truncations of the N- or C-terminus orboth termini and/or amino acid substitutions, deletions or additionswherein the variants retain superior mitogenic activity mediated viaFGFRs with unimpaired or improved affinities compared to the wild typeparent FGF-2, from which it was derived. The additional modificationsfunction to improve certain properties of the variants includingenhanced stability, increased yield of recombinants, solubility andother properties known in the art. For example, FGF-2 may comprise aminoacid substitutions at amino acid positions 3 and 5 wherein alanine (Ala,A) and serine (Ser, S) are replaced with Glutamine (Gln, Q) (A3Q andS5Q) providing variants with improved yields and stability. A currentlypreferred embodiment of the present invention is denoted hereinFGF2(3,5Q)-N111X, SEQ ID NO:4. Table 2 presents a summary of receptorspecificity of the FGF2 variants of the present invention.

TABLE 2 Specificity of FGF2(3,5Q)-N111X variants towards FGFR-expressingFDCP cells. Mutant FGFR1 FGFR2 FGFR3IIIb FGFR3IIIc FGF-2 + + − +FGF2(3,5Q)-N111G ++ + − +++ FGF2-N111R +++ + − +

The corresponding position of N111 in FGF-4 is N165 (numbering accordingto the 206 aa form). FGF-4 was shown to have high affinity for the HSPGswhich enhances FGFR binding and activation. The wild type FGF-4 is shownto induce a high level of proliferation through FGFR1 and a lower levelthrough FGFR2, with negligible activity through FGFR3. Activity throughFGFR3, as measured in a proliferation assay, is enhanced by substitutionof an amino acid at position N165 and truncation of N-terminal aminoacids. A currently preferred embodiment of the present invention is anFGF4 variant denoted herein FGF4-N165X, having SEQ ID NO:6, wherein X isother than asparagine. A currently more preferred embodiment of thepresent invention is denoted herein L55M-FGF4-N165X, SEQ ID NO:7,wherein X is other than asparagine. This variant induces proliferationthrough FGFR3 while maintaining the same level of activity through FGFR1and FGFR2.

The therapeutic utility of these novel FGF-2 and FGF-4 variants isdisclosed for both normal and abnormal FGF receptors, including but notlimited to cartilage and bone regeneration and bone fracture healing,articular chondrocyte proliferation, osteoporosis, wound healing,ischemic tissue repair, neural tissue survival and repair andneovascularization. Additionally, the high receptor-specificity of thesenovel variants warrants their use in targeting bioactive agents, inparticular cytotoxic material to cells overexpressing FGFR receptors,for the treatment of proliferative diseases.

In a currently preferred embodiment of the present invention thevariants having SEQ ID NOS:1-9 are formulated to provide pharmaceuticalcompositions useful for promoting or accelerating repair or regenerationof endochondral bone, intramembranous bone, cartilage, includingarticular cartilage, spinal defects and other skeletal disorders and forpromoting or accelerating neovascularization in indications includingburns, cuts, lacerations, bed sores, ulcers such as those seen indiabetic patients, repair and regeneration of tissue, includingskeletal, skin and vascular tissue. The compositions comprise thevariant and further comprise an HSPG as carrier or stabilizer and amatrix-free or matrix device.

Unexpectedly, certain FGF variants of the present invention were foundto retain binding affinity to specific FGF receptors while exhibitingreduced receptor-mediated biological activity, providing variants usefulfor targeting bioactive agents including polypeptides, peptides andanalogs and drugs to specific tissue. Effectively, the variantpolypeptides are useful as carriers which can be used for site-specificdelivery and concentration of bioactive agent to cells, tissues, ororgans in which a therapeutic effect is desired to be effected.

The equivalent position of N111 in FGF-9 is W144 (tryptophan at position144 of the wild type protein). We generated substitutions at the W144site and tested them for receptor specificity. The tryptophan wasreplaced with either Gly (G), Ala (A), Val (V), Asn (N), Glu (E) or Arg(R). The W144G, W144V, W144E and W144R variants showed diminishedspecificity towards FGFR1 and retention of specificity towards the FGFR3receptor. The W144A or W144N variants behaved as native FGF-9. Inaddition, a substitution of the adjacent Asn (asparagine) at position143 to a Ser (Serine), N143S, resulted in activation of FGFR3 and notFGFR1. Table 3 summarizes the specificity of the FGF-9 variants to FDCPcells transfected with the various FGFR as determined in a cellproliferation assay.

TABLE 3 Specificity of FGF9 variants towards FGFR-expressing FDCP cells.Mutant FGFR-1 FGFR-3 WT-FGF-9 + + FGF-9 W144G − + FGF-9 W144A + + FGF-9W144V − + FGF-9 W144N + + FGF-9 W144E − + FGF-9 W144R − + FGF-9 N143S −+

According to additional preferred embodiments, the FGF comprises thesubstitution of Trp 144 (W144) of FGF-9 with either Gly (G), Val (V),Glu (E) or Arg (R).

In a preferred embodiment of the present invention, the variantcomprises one or more amino acid substitutions in the β8-β9 loop and atruncation at either or both the N or C terminus. These variants wouldbe advantageous in terms of their stability and/or solubility andreceptor affinity and specificity, and concomitant reduced biologicalactivity. FIG. 5B shows the reduced level of mitogenic activity ofcertain preferred FGF-9 variants in a proliferation assay in FGFR1 orFGFR3-transfected FDCP cells. The X axis is concentration of FGF9variant measured in ng/ml, while the Y axis depicts absorbtion at 490 nmand reflects mitogenicity. An inactive variant will elicit a mitogenicresponse through a specific receptor at a level not to be lower thanatleast half two-fold of that of the corresponding native FGF at aconcentration not higher than 50-fold of that of the native FGF, morepreferably not higher than 20-fold and most preferably not higher than10-fold than that of the native FGF receptor ligand.

Upon removal of amino acid residues near and into the core structure,the FGF protein loses receptor affinity. FGF9-2, a 127 aa represented asSEQ ID NO: 12 has reduced mitogenic capacity relative to wild typeFGF-9. The R64M-FGF9 variant of 145 aa, represented as SEQ ID NO:10,provides the shortest FGF-9 polypeptide that retains binding specificitytoward FGFR3 and has lost the binding capacity toward FGFR1, asdetermined in a mitogenic assay. FIG. 5B shows that although themitogenic activity of R64M-FGF9 is reduced in comparison to that of wildtype FGF-9 the variant retains high specificity towards FGFR3 yet showsreduced activity and does not elicit a response through FGFR1. Acurrently more preferred embodiment of the invention is an R64M-FGF9variant further comprising an amino acid substitution in the β8-β9 loop.These variants are denotedherein R64M-FGF9-W144X and R64M-FGF9-N143X,SEQ ID NO:11 and 14, respectively. Corresponding polynucleotidesequences are represented as SEQ ID NOS:26 and 29, respectively.

A currently most re preferred embodiment is the inactive variants ofFGF-9 denoted herein as FGF9-2-W144X, the amino acid sequence of whichis represented as SEQ ID NO:12 wherein X is other than tryptophan andcurrently most preferred amino acid substitution I selected from Glycine(G), Arg (R), Val (V) or Glu (E). The corresponding polynucleotidesequence is presented as SEQ ID NO:28. The currently more preferredembodiments of the inactive variant of FGF-9 are denoted herein asFGF9-2-W144G, FGF9-2-W144V and FGF9-2-W144E. Introduction of glycine atposition 144 of FGF-9 abolished its binding towards FGFR1 whileretaining significant affinity towards FGFR3 and to a lesser extent,FGFR2. Furthermore, the FGF9-2-W144G variant specifically targets thegrowth plate, as shown in FIGS. 7A and 7B.

Methods of Producing and Using Variants

The most preferred method for producing the variants is throughrecombinant DNA technologies, well known to those skilled in the art.For example, the variants may be prepared by Polymerase Chain Reaction(PCR) using specific primers for each of the truncated forms or theamino acid substitutions as disclosed herein below. The PCR fragmentsmay be purified on an agarose gel and the purified DNA fragment may becloned into an expression vector and transfected into host cells. Thehost cells may be cultured and the protein harvested according tomethods known in the art. According to another aspect of the presentinvention it is disclosed that the preferred variant FGFs have improvedtherapeutic utility in diseases and disorders involving FGF receptors.

The therapeutic utility of these novel variants is disclosed for bothnormal and abnormal FGF receptors, including but not limited to boneregeneration and bone fracture healing, osteoporosis, wound healing,malignant cells overexpressing FGFR receptors, Achondroplasia andHypochondroplasia (a condition associated with moderate but variable,disproportionate shortness of limbs). According to currently morepreferred embodiments it is possible to target drugs and other bioactivemolecules, including but not limited to cytotoxic drugs, peptides andanalogs and polypeptides to cells bearing FGFR3 without appreciablyaffecting cells bearing FGFR1. This is accomplished by conjugating thedrug of choice to a variant FGF of the invention. According to even morepreferred embodiments of the present invention it is now possible totarget drugs and other bioactive molecules, including but not limited topeptides and cytotoxic drugs, to one or more specific subtype of FGFR2and/or FGFR3. Most preferred embodiments of the invention areparticularly useful in conjugates with drugs for inhibiting cellproliferation and facilitating or enhancing the treatment of defects ortumors bearing a specific receptor subtype, without interfering with thegrowth of normal cells or tissues bearing other receptor subtypes. In anon-limiting example, FGF9-2-W144G targeting compositions can comprise aFGF9-2-W144G component and cytotoxin that are covalently bound to eachother. Another example is a conjugate with a tyrosine inhibitor such as,but not limited to, genistein. Alternatively, FGF9-W144G targetingcompositions can comprise an FGF9-2-W144G targeting fusion protein. In acurrently most preferred embodiment a fusion protein of an inactivevariant of the present invention and a peptide or peptide analog is usedfor targeting of said peptide or analog to a specific cell, tissue ororgan.

A “targeting molecule” is defined herein as a molecule which is bound bya receptor and transported to a cell by a receptor-mediated process.Examples of suitable targeting molecules include, but are not limitedto, glucose, galactose, mannose, insulin, a peptide growth factor,cobalamin, folic acid or derivatives, biotin or derivatives, albumin,texaphyrin, metallotexaphyrin, porphyrin, any vitamin, any coenzyme, anantibody, an antibody fragment (e.g., Fab) and a single chain antibodyvariable region (scFv). A skilled artisan will readily recognize othertargeting molecules including ligands which bind to cell receptors andwhich are transported into a cell by a receptor-mediated process. Thepresent invention is intended to include all such targeting molecules.

In another currently preferred embodiment bioactive agents are targetedto a desired tissue, specifically the growth plate of the bones. Thismay be achieved by methods known to one skilled in the art and include,in a nonlimiting manner, a chimeric protein comprising a FGF variant ofthe present invention as carrier fused to a bioactive agent includingpeptides and petide analogs. According to a currently more preferredembodiment a natriuretic peptide or a functional derivative thereof isfused to an FGF variant of the invention, preferably FGF9-2-W144G,herein denoted FGF9-2-W144G-CNP having SEQ ID NO:16, whereinFGF9-2-W144G is fused to CNP via a Glycine linker. Alternatively, theCNP moiety is linked to an FGF variant, such that the CNP moiety lies 5′to the FGF, herein denoted CNP(1-22)-FGF9-2-W144G, having SEQ ID NO:17.

According to the principles of the present invention it is now disclosedthat the through introduction of a single amino acid substitution withinthe β8-β9 loop, an FGF may undergo interconversion from a mitogen to adifferentiation factor, or from a differentiation factor to a mitogen.This property of the novel variants warrants their use in selectivelyinducing proliferation and differentiation of various cell types. Forare more potent inducers of proliferation than the native FGF2.Alternatively, the FGF9 variants, W144G-FGF9 and L37M-W144G-FGF9, havingSEQ ID NO:8 and 9 respectively, induce differentiation of articularchondrocytes whereas the wild type protein FGF-9 is both a weak mitogenand a weak differentiation factor. The variants of the present inventionmay be used in vitro or in vivo, alone or in combination to achieve adesired effect of proliferation and/or differentiation. In onenon-limiting example of autologous chondrocyte implantation (ACI) theFGF2-N111X variant is added to a culture of human chondrocytes preparedfrom a biopsy to induce rapid proliferation of the cells. This isfollowed by the addition of the FGF9-W144G variant to inducedifferentiation of the cultured cells. The differentiated cells may thenbe reintroduced to a subject in need of treatment for the repair ofdiseased or traumatized cartilage tissue. The variants may be used toculture a variety of cell types including osteoblasts, neurons,hematopoietic cells, progenitor cells and stem cells. Furthermore, thevariants may be used for the induction of proliferation and/ordifferentiation in vivo.

Pharmacology

The present invention also contemplates pharmaceutical formulations,both for veterinary and for human medical use, which comprise as theactive agent one or more polypeptide(s) of the invention, as well as theuse of a polypeptide of the invention in the manufacture of a medicamentfor the treatment or prophylaxis of the conditions variously describedherein.

In such pharmaceutical and medicament formulations, the active agentpreferably is utilized together with one or more pharmaceuticallyacceptable carrier(s) therefore and optionally any other therapeuticingredients. The carrier(s) must be pharmaceutically acceptable in thesense of being compatible with the other ingredients of the formulationand not unduly deleterious to the recipient thereof. The active agent isprovided in an amount effective to achieve the desired pharmacologicaleffect, as described above, and in a quantity appropriate to achieve thedesired daily dose.

Another critically functional component in FGF signaling isproteoglycans such as heparan sulfate. FGFs fail to bind and activateFGF receptors in cells deprived of endogenous heparan sulfate.Proteoglycan refers to heparan sulfate proteoglycans (HSPG) or othertypes including chondroitin sulfate-, keratin sulfate-, and dermatansulfate proteoglycans.

The dose of the pharmaceutical composition of the present invention mayvary with the kind of disease, the age of patient, body weight, theseverity of disease, the route of administration, etc.

Apart from other considerations, the fact that the novel activeingredients of the invention are polypeptides, polypeptide variants orfusion proteins dictates that the formulation be suitable for deliveryof these types of compounds. Clearly, peptides are less suitable fororal administration due to susceptibility to digestion by gastric acidsor intestinal enzymes. Apart from other considerations, the fact thatthe novel active ingredients of the invention are polypeptides dictatesthat the formulation be suitable for delivery of this type of compounds.Clearly, peptides are less suitable for oral administration due tosusceptibility to digestion by gastric acids or intestinal enzymes.Specific formulations may be designed to circumvent these problems,including enterocoating, gelatin capsules, emulsions and the like.Nevertheless, bioavailability is impaired by poor gastrointestinalabsorption and the routes of administration are preferably parenteral.The preferred routes of administration are intra-articular (IA),intravenous (IV), intramuscular (IM), subcutaneous (SC), intradermal(ID), or intrathecal (IT). A more preferred route is by direct injectionat or near the site of disorder or disease.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active variant selected from the sequences, SEQ IDNO:1-17 described herein, or physiologically acceptable salts orprodrugs thereof, with other chemical components such as physiologicallysuitable carriers and excipients. The purpose of a pharmaceuticalcomposition is to facilitate administration of a compound to anorganism.

The term “prodrug” refers to an agent, which is converted into an activeparent drug in vivo. Prodrugs are often useful because in some instancesthey may be easier to administer than the parent drug. They may, forinstance, be bioavailable by oral administration whereas the parent drugis not. The prodrug may also have improved solubility compared to theparent drug in pharmaceutical compositions.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of acompound. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.Pharmaceutical compositions may also include one or more additionalactive ingredients.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, grinding, pulverizing, dragee-making,levigating, emulsifying, encapsulating, entrapping or lyophilizingprocesses.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active compounds intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the compounds of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hank's solution, Ringer's solution, or physiological saline buffer.For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants for exampleDMSO, or polyethylene glycol are known in the art.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive compounds may be dissolved or suspended in suitable liquids, suchas fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration. For buccal administration, the compositions may take theform of tablets or lozenges formulated in conventional manner. Foradministration by inhalation, the variants for use according to thepresent invention are delivered in the form of an aerosol spraypresentation from a pressurized pack or a nebulizer with the use of asuitable propellant known in the art. In the case of a pressurizedaerosol, the dosage unit may be determined by providing a valve todeliver a metered amount. Capsules and cartridges of, e.g., gelatin foruse in an inhaler or insufflator may be formulated containing a powdermix of the peptide and a suitable powder base.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active ingredients in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidsesters such as ethyl oleate, triglycerides or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, including but not limited to naturalsubstances and polymers such as collagen, sorbitol, dextran orhyaluronic acid (HA) and derivatives, synthetic polymers, cellulosederivatives including sodium carboxymethyl cellulose (CMC) andderivatives of said substances or any natural or synthetic carrier knownin the art (Pillai and Panchagnula, Curr. Opin. Chem. Biol. 5, 447,2001). Optionally, the suspension may also contain suitable stabilizersor agents, which increase the solubility or stability of the compounds,to allow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forreconstitution with a suitable vehicle, e.g., sterile, pyrogen-freewater, before use.

The compounds of the present invention may also be formulated in rectalcompositions such as suppositories or retention enemas, using, e.g.,conventional suppository bases.

The formulations of the active variants may be administered topically asa gel, ointment, cream, emulsion or sustained release formulationincluding a transdermal patch. The pharmaceutical compositions hereindescribed may also comprise suitable solid of gel phase carriers orexcipients. Examples of such carriers or excipients include, but are notlimited to, calcium carbonate, calcium phosphate, various sugars,starches, cellulose derivatives, gelatin and polymers such aspolyethylene glycols.

For treating bone or other tissue, for example bone fractures, cartilagedefects or tissue repair, administration may be preferred locally bymeans of a direct injection at or near the site of target or by means ofa subcutaneous implant, staples or slow release formulation implanted ator near the target. Suitable devices for direct injection orimplantation are biocompatable and maybe matrix-free or comprise amatrix. Matrix-free devices include, in a non-limiting manner, amorphousmaterials formulated as a paste, putty, viscous liquid or gel. In oneembodiment of the present invention formulations of the variant comprisematrix-free devices including Pluronic poloxamers orcarboxymethylcellulose (U.S. Pat. No. 6,281,195), polysaccharides orcross-linked polysaccharides (U.S. Pat. No. 6,303,585) and hyaluronicacid (U.S. Pat. No. 6,221,854).

A matrix affords a certain structural component providing a permanent ortemporary scaffold for infiltrating cells. It may alternatively providea scaffold for administration of a variant of the invention to thetissue in need thereof. Release of the variant may controlled. Matricesinclude, in a non-limiting manner, include collagen compositions (WO00/47114; U.S. Pat. Nos. 4,394,370 and 5,425,769), polymeric andcopolymeric compositions (U.S. Pat. No. 5,650,180), calcium phosphateparticle and ceramic compositions (U.S. Pat. No. 6,231,607), includinghydroxyapatite compositions (WO 90/01342 and U.S. Pat. Nos. 5,338,772and 4,795,467), coral, gelatins and demineralized bone. Furthermore, amatrix may be in the form of an implant, a single layer or multilayeredcomposition, sheet, or in particulate form.

A pharmaceutical composition comprising as an active ingredient avariant of the invention in a matrix or matrix-free device may furthercomprise stabilizers including heparin sulfate or other HSPGs orcarriers as those listed above.

A currently preferred embodiment of the present invention provides amethod of administering an FGF variant of the present invention, havingSEQ ID Nos: 1-7, in combination with a calcium phosphate based matrix toa patient in need thereof.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount of acompound effective to prevent, alleviate or ameliorate symptoms of adisease of the subject being treated. Determination of a therapeuticallyeffective amount is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

Toxicity and therapeutic efficacy of the peptides described herein canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., by determining the IC₅₀ (the concentrationwhich provides 50% inhibition) and the LD₅₀ (lethal dose causing deathin 50% of the tested animals) for a subject compound. The data obtainedfrom these cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. Depending on the severity and responsiveness of the conditionto be treated, dosing can also be a single administration of a slowrelease composition, with course of treatment lasting from several daysto several weeks or until cure is effected or diminution of the diseasestate is achieved. The amount of a composition to be administered willbe dependent on the subject being treated, the severity of theaffliction, the manner of administration, the judgment of theprescribing physician, and other factors.

The following example is an illustration only of a method of treating asubject with a variant according to the invention, in order to treat apathological condition associated with tissue trauma or a relatedcondition, and is not intended to be limiting.

The method includes the step of administering the variant or chimera orfusion protein, in a pharmaceutically acceptable carrier as describedabove, to a subject to be treated. The medicament is administeredaccording to an effective dosing methodology, preferably until apredefined endpoint is reached, such as a reduction or amelioration ofthe pathological condition in the subject.

The present invention also relates to methods of treatment of thevarious pathological conditions described above, by administering to apatient a therapeutically effective amount of the compositions of thepresent invention. The term administration as used herein encompassesoral, parenteral, intravenous, intramuscular, subcutaneous, transdermal,intrathecal, rectal and intranasal administration.

The present invention further relates to method for the use of theactive FGF variants to prepare medicaments useful in inducing boneformation and fracture healing as well as in the detection and treatmentof various FGFR-related disorders including skeletal disorders such asachondroplasia and thanatophoric dysplasia and certain types of cancerincluding but not limited to transitional cell carcinoma (TCC) of thebladder, multiple myeloma, chronic myeloid leukemia (CML) and cervicalcarcinoma.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification. In addition,citation or identification of any reference in this application shallnot be construed as an admission that such reference is available asprior art to the present invention.

The subsequent sequences are preferred embodiments according to theinvention. Sequences listed are according to the 155 amino acid isoformof human FGF-2. Those skilled in the art will recognize that thepolynucleotide sequences disclosed in SEQ ID NOs:18-32 represent asingle allele of the human FGF-2, FGF-4 and FGF-9 genes andpolypeptides, and that allelic variation are expected to occur. Allelicvariants can be cloned by probing cDNA or genomic libraries or begenerated by PCR from total RNA, cDNA or genomic DNA from differentindividuals according to standard procedures. Allelic variants of thepolynucleotide sequence, including those containing silent mutations andthose in which mutations result in amino acid sequence changes, arewithin the scope of the present invention.

The 18 kDa FGF-2 molecule is 155 aa in length when translated from anAUG (methionine) start codon (Abraham et al. EMBO J. 5, 2523, 1986). Inaddition, there are at least four alternate start codons (CUG, Leu) thatprovide N-terminal extensions of 41, 46, 55, or 133 aa, resulting inproteins of 22 kDa (196 aa), 22.5 kDa (201 aa), 24 kDa (210 aa) and 34kDa (288 aa), respectively, having the potential to perform the samefunction (reviewed in Okada-Ban et al., Int J Biochem Cell Biol, 32,263, 2000).

The core of approximately 120 amino acids of FGF (amino acids 66-190 ofFGF-9, amino acids 30-152 of the 155 aa isoform of FGF-2) has been shownto be crucial for FGF function. Truncations extending within a few aminoacids near to or into the core result in reduced biological activity, asdetermined by proliferation assays. It is now disclosed that FGFvariants with reduced biological activity are useful for targetingbioactive agents to specific tissues.

Sequences

The amino acid sequences of the preferred embodiments of the presentinvention are disclosed as follows:

Amino Acid Sequence of FGF2-N111X  (SEQ ID NO: 1)MAAGSITTLP ALPEDGGSGA FPPGHFKDPK RLYCKNGGFFLRIHPDGRVD GVREKSDPHI KLQLQAEERG VVSIKGVCANRYLAMKEDGR LLASKCVTDE CFFFERLESN  X YNTYRSRKYTSWYVALKRT GQYKLGSKTG PGQKAILFLP MSAKSwherein X is other than N and more preferably selected from R or G.Amino Acid Sequence of FGF2-N111R  (SEQ ID NO: 2)MAAGSITTLP ALPEDGGSGA FPPGHFKDPK RLYCKNGGFFLRIHPDGRVD GVREKSDPHI KLQLQAEERG VVSIKGVCANRYLAMKEDGR LLASKCVTDE CFFFERLESN RYNTYRSRKYTSWYVALKRT GQYKLGSKTG PGQKAILFLP MSAKSAmino Acid Sequence of FGF2-N111G  (SEQ ID NO: 3)MAAGSITTLP ALPEDGGSGA FPPGHFKDPK RLYCKNGGFFLRIHPDGRVD GVREKSDPHI KLQLQAEERG VVSIKGVCANRYLAMKEDGR LLASKCVTDE CFFFERLESN  G YNTYRSRKYTSWYVALKRT GQYKLGSKTG PGQKAILFLP MSAKSAmino Acid Sequence of FGF2(3, 5Q)-N111X  (SEQ ID NO: 4) MA Q G QITTLP ALPEDGGSGA FPPGHFKDPK RLYCKNGGFFLRIHPDGRVD GVREKSDPHI KLQLQAEERG VVSIKGVCANRYLAMKEDGR LLASKCVTDE CFFFERLESN  X YNTYRSRKYTSWYVALKRT GQYKLGSKTG PGQKAILFLP MSAKSwherein X is other than N and more preferably selected from G or R.Amino Acid Sequence of FGF2(3, 5Q)-N111G  (SEQ ID NO: 5) MA Q G QITTLP ALPEDGGSGA FPPGHFKDPK RLYCKNGGFFLRIHPDGRVD GVREKSDPHI KLQLQAEERG VVSIKGVCANRYLAMKEDGR LLASKCVTDE CFFFERLESN  G YNTYRSRKYTSWYVALKRT GQYKLGSKTG PGQKAILFLP MSAKSAmino Acid sequence of human FGF4-N165X 206 aa (SEQ ID NO: 6)MSGPGTAAVA LLPAVLLALL APWAGRGGAA APTAPNGTLEAELERRWESL VALSLARLPV AAQPKEAAVQ SGAGDYLLGIKRLRRLYCNV GIGFHLQALP DGRIGGAHAD TRDSLLELSPVERGVVSIFG VASRFFVAMS SKGKLYGSPF FTDECTFKEI LLPN XYNAYE SYKYPGMFIA LSKNGKTKKG NRVSPTMKVT HFLPRLwherein X is other than N and more preferably G.Amino acid sequence of human L55M-FGF4-N165X 152 AA  (SEQ ID NO: 7)     M ARLPV AAQPKEAAVQ SGAGDYLLGI KRLRRLYCNVGIGFHLQALP DGRIGGAHAD TRDSLLELSP VERGVVSIFGVASRFFVAMS SKGKLYGSPF FTDECTFKEI LLPN X YNAYESYKYPGMFIA LSKNGKTKKG NRVSPTMKVT HFLPRLwherein X is other than N and more preferably G.Amino acid sequence of human W144X-FGF9 208 AA (SEQ ID NO:8) disclosedin PCT patent application WO 02/36732:

Amino acid sequence of human W144X-FGF9 208 AAdisclosed in PCT patent application WO 02/36732: (SEQ ID NO: 8)MAPLGEVGNY FGVQDAVPFG NVPVLPVDSP VLLSDHLGQSEAGGLPRGPA VTDLDHLKGI LRRRQLYCRT GFHLEIFPNGTIQGTRKDHS RFGILEFISI AVGLVSIRGV DSGLYLGMNE KGELYGSEKL TQECVFREQF EEN XYNTYSS NLYKHVDTGR RYYVALNKDG TPREGTRTKR HQKFTHFLPR PVDPDKVPEL YKDILSQSwherein X is other than W and more preferablyselected from G, R, E or V.Aminod Acid sequence of L37M-W144X-FGF9 172aa disclosed in PCT patent application WO 02/36732: (SEQ ID NO: 9) MGQSEAGGLP RGPAVTDLDH LKGILRRRQL YCRTGRHLEIFPNGTIQGTR KDHSRFGILE FISIAVGLVS IRGVDSGLYLGMNEKGELYG SEKLIQECVF REQFEEN X YN IYSSNLYKHVDIGRRYYVAL NKDGIPREGI RIKRHQKFIH FLPRPVDPDK VPELYKDILS QSwherein X is other than W and more preferablyselected from G, R, E or V. Amino Acid sequence of R64M-FGF9 145 aadisclosed in PCT patent application WO 02/36732: (SEQ ID NO: 10)MQLYCRTGFH LEIFPNGTIQ GTRKDHSRFG ILEFISIAVGLVSIRGVDSG LYLGMNEKGE LYGSEKLTQE CVFREQFEENWYNTYSSNLY KHVDTGRRYY VALNKDGTPR EGTRTKRHQK FTHFLPRPVD PDKVPELYKD ILSQSwherein X is other than W and more preferablyselected from G, R, E or V.Amino Acid sequence of R64M-FGF9-W144X 145 aa (SEQ ID NO: 11)MQLYCRTGFH LEIFPNGTIQ GTRKDHSRFG ILEFISIAVGLVSIRGVDSG LYLGMNEKGE LYGSEKLTQE CVFREQFEEN XYNTYSSNLY KHVDTGRRYY VALNKDGTPR EGTRTKRHQK FTHFLPRPVD PDKVPELYKD ILSQSwherein X is other than W and more preferablyselected from G, R, E or V. Amino Acid sequence of FGF9-2 127 aadisclosed in PCT patent application WO 02/36732: (SEQ ID NO: 12)MQLYCRTGFH LEIFPNGTIQ GTRKDHSRFG ILEFISIAVGLVSIRGVDSG LYLGMNEKGE LYGSEKLTQE CVFREQFEENWYNTYSSNLY KHVDTGRRYY VALNKDGTPR EGTRTKRHQK FTHFLPRAmino Acid sequence of FGF9-2-W144X 127 aa (SEQ ID NO: 13)MQLYCRTGFH LEIFPNGTIQ GTRKDHSRFG ILEFISIAVGLVSIRGVDSG LYLGMNEKGE LYGSEKLTQE CVFREQFEEN XYNTYSSNLY KHVDTGRRYY VALNKDGTPR EGTRTKRHQK FTHFLPRwherein X is other than W and more preferablyselected from G, R, E or V.Amino Acid sequence of R64M-FGF9-N143X 145 aa (SEQ ID NO: 14)MQLYCRTGFH LEIFPNGIIQ GTRKDHSRFG ILEFISIAVGLVSIRGVDSG LYLGMNEKGE LYGSEKLTQE CVFREQFEE XWYNTYSSNLY KHVDTGRRYY VALNKDGTPR EGTRTKRHQK FTHFLPRPVD PDKVPELYKD ILSQSwherein X is other than N and more preferably S.Amino Acid sequence of FGF9-2-N143X 127 aa (SEQ ID NO: 15)MQLYCRTGFH LEIFPNGTIQ GTRKDHSRFG ILEFISIAVGLVSIRGVDSG LYLGMNEKGE LYGSEKLTQE CVFREQFEE XWYNTYSSNLY KHVDTGRRYY VALNKDGTPR EGTRTKRHQK FTHFLPRwherein X is other than N and more preferably S.Amino Acid sequence of FGF9-2-W144X-CNP(1-22) (SEQ ID NO: 16)MQLYCRTGFH LEIFPNGTIQ GTRKDHSRFG ILEFISIAVGLVSIRGVDSG LYLGMNEKGE LYGSEKLTQE CVFREQFEEN XYNTYSSNLY KHVDTGRRYY VALNKDGTPR EGTRTKRHQKFTHFLPRGGG GLSKGCFGLK LDRIGSMSGL GCwherein X is other than W and more preferablyselected from G, R, E or V.Amino Acid sequence of CNP(1-22)-FGF9-2-W144X (SEQ ID NO: 17)MGLSKGCFGL KLDRIGSMSG LGCGGGGGGG GQLYCRTGFHLEIFPNGTIQ GTRKDHSRFG ILEFISIAVG LVSIRGVDSGLYLGMNEKGE LYGSEKLTQE CVFREQFEEN  X YNTYSSNLYKHVDTGRRYY VALNKDGTPR EGTRTKRHQK FTHFLPR

The corresponding polynucleotide sequences are as follows:

AGGATGGCGG CAGCGGCGCC TTCCCGCCCG GGCACTTCAAGGACCCCAAG CGGCTGTACT GCAAAAACGG GGGCTTCTTCCTGCGCATCC ACCCCGACGG CCGAGTTGAC GGGGTCCGGGAGAAGAGCGA CCCTCACATC AAGCTACAAC TTCAAGCAGAAGAGAGAGGA GTTGTGTCTA TCAAAGGAGT GTGTGCTAACCGGTACCTGG CTATGAAGGA AGAGAGAGGA TTACTGGCTTCTAAATGTGT TACGGATGAG TGTTTCTTTT TTGAACGATT GGAATCTAAT  NNNTACAATA CTTACCGGTC TAGAAAATACACCAGTTGGT ATGTGGCATT GAAACGAACT GGGCAGTATAAACTTGGTTC CAAAACAGGA CCTGGGCAGA AAGCTATACTTTTTCTTCCA ATGTCTGCTA AGAGCTGAwherein NNN is other than a codon coding for Asn(AAT or AAC) or a stop codon more preferablya codon coding for amino acid Gly or Arg.wherein NNN is other than a codon coding for Asn (AAT or AAC) or a stopcodon and is more preferably a codon coding for amino acid Gly or Arg.

Sequence of FGF2-N111G DNA (SEQ ID NO: 19)ATGGCTGCCG GGAGCATCAC CACGCTGCCC GCCCTTCCGGAGGATGGCGG CAGCGGCGCC TTCCCGCCCG GGCACTTCAAGGACCCCAAG CGGCTGTACT GCAAAAACGG GGGCTTCTTCCTGCGCATCC ACCCCGACGG CCGAGTTGAC GGGGTCCGGGAGAAGAGCGA CCCTCACATC AAGCTACAAC TTCAAGCAGAAGAGAGAGGA GTTGTGTCTA TCAAAGGAGT GTGTGCTAACCGGTACCTGG CTATGAAGGA AGATGGAAGA TTACTGGCTTCTAAATGTGT TACGGATGAG TGTTTCTTTT TTGAACGATT GGAATCTAAT  NNNTACAATA CTTACCGGTC TAGAAAATACACCAGTTGGT ATGTGGCATT GAAACGAACT GGGCAGTATAAACTTGGTTC CAAAACAGGA CCTGGGCAGA AAGCTATACTTTTTCTTCCA ATGTCTGCTA AGAGCTGAwherein NNN is a codon coding for amino acid Gly (GGT, GGC, GGA, GGG).Sequence of FGF2-N111R DNA (SEQ ID NO: 20)ATGGCTGCCG GGAGCATCAC CACGCTGCCC GCCCTTCCGGAGGATGGCGG CAGCGGCGCC TTCCCGCCCG GGCACTTCAAGGACCCCAAG CGGCTGTACT GCAAAAACGG GGGCTTCTTCCTGCGCATCC ACCCCGACGG CCGAGTTGAC GGGGTCCGGGAGAAGAGCGA CCCTCACATC AAGCTACAAC TTCAAGCAGAAGAGAGAGGA GTTGTGTCTA TCAAAGGAGT GTGTGCTAACCGGTACCTGG CTATGAAGGA AGATGGAAGA TTACTGGCTTCTAAATGTGT TACGGATGAG TGTTTCTTTT TTGAACGATT GGAATCTAAT CG NTACAATA CTTACCGGTC TAGAAAATACACCAGTTGGT ATGTGGCATT GAAACGAACT GGGCAGTATAAACTTGGTTC CAAAACAGGA CCTGGGCAGA AAGCTATACTTTTTCTTCCA ATGTCTGCTA AGAGCTGA wherein N is selected from A, C, G or TSequence of FGF2(3Q5Q)-N111X DNA (SEQ ID NO: 21) ATGGCT CAX G GG CAXATCAC CACGCTGCCC GCCCTTCCGG AGGATGGCGG CAGCGGCGCC TTCCCGCCCG GGCACTTCAAGGACCCCAAG CGGCTGTACT GCAAAAACGG GGGCTTCTTCCTGCGCATCC ACCCCGACGG CCGAGTTGAC GGGGTCCGGGAGAAGAGCGA CCCTCACATC AAGCTACAAC TTCAAGCAGAAGAGAGAGGA GTTGTGTCTA TCAAAGGAGT GTGTGCTAACCGGTACCTGG CTATGAAGGA AGATGGAAGA TTACTGGCTTCTAAATGTGT TACGGATGAG TGTTTCTTTT TTGAACGATT GGAATCTAAT  NNNTACAATA CTTACCGGTC TAGAAAATACACCAGTTGGT ATGTGGCATT GAAACGAACT GGGCAGTATAAACTTGGTTC CAAAACAGGA CCTGGGCAGA AAGCTATACTTTTTCTTCCA ATGTCTGCTA AGAGCTGAwherein nuleotides 9 and 15 are independentlychosen from A or G and the codon encoded by NNNAT POSITION 331-333 is other than a codon codingfor Asn (AAT or AAC) or a stop codon and is morepreferably encodes for amino acid Gly or Arg.

Sequence of FGF2(3Q5Q)-N111G DNA (SEQ ID NO: 22) ATGGCT CAX G GG CAXATCAC CACGCTGCCC GCCCTTCCGG AGGATGGCGG CAGCGGCGCC TTCCCGCCCG GGCACTTCAAGGACCCCAAG CGGCTGTACT GCAAAAACGG GGGCTTCTTCCTGCGCATCC ACCCCGACGG CCGAGTTGAC GGGGTCCGGGAGAAGAGCGA CCCTCACATC AAGCTACAAC TTCAAGCAGAAGAGAGAGGA GTTGTGTCTA TCAAAGGAGT GTGTGCTAACCGGTACCTGG CTATGAAGGA AGAGAGAGGA TTACTGGCTTCTAAATGTGT TACGGATGAG TGTTTCTTTT TTGAACGATT GGAATCTAAT  GGNTACAATA CTTACCGGTC TAGAAAATACACCAGTTGGT ATGTGGCATT GAAACGAACT GGGCAGTATAAACTTGGTTC CAAAACAGGA CCTGGGCAGA AAGCTATACTTTTTCTTCCA ATGTCTGCTA AGAGCTGAwherein nuleotides 9 and 15 are independentlychosen from A or G and the N at position 333is selected from A, C, G or T.

Sequence of FGF4-N165X DNA (SEQ ID NO: 23)ATGTCGGGGC CCGGGACGGC CGCGGTAGCG CTGCTCCCGGCGGTCCTGCT GGCCTTGCTG GCGCCCTGGG CGGGCCGAGGGGGCGCCGCC GCACCCACTG CACCCAACGG CACGCTGGAGGCCGAGCTGG AGCGGCGCCG GGAGAGCCTG GTGGCGCTCTCGTTGGCGCG CCTGCCGGTG GCAGCGCAGC CCAAGGAGGCGGCCGTCCAG AGCGGCGCCG GCGACTACCT GCTGGGCATCAAGCGGCTGC GGCGGCTCTA CTGCAACGTG GGCATCGGCTTCCACCTCCA GGCGCTCCCC GACGGCCGCA TCGGCGGCGCGCACGCGGAC ACCCGCGACA GCCTGCTGGA GCTCTCGCCCGTGGAGCGGG GCGTGGTGAG CATCTTCGGC GGGCGCCGCCGGTTCTTCGT GGCCATGAGC AGCAAGGGCA AGCTCTATGGCTCGCCCTTC TTCACCGATG AGTGCACGTT CAAGGAGATT CTCCTTCCCA AC XXXTACAA CGCCTACGAG TCCTACAAGT ACCCCGGCAT GTTCATCGCC CTGAGCAAGA ATGGGAAGACCAAGAAGGGG AACCGAGTGT CGCCCACCAT GAAGGTCACC CACTTCCTCC CCAGGCTGwherein XXX is other than a codon coding forAsn (AAT or AAC) or a stop codon and is morepreferably ecodes for amino Gly (GGA, GGC, GGG, GGT).

Sequence of L55M-FGF4-N165X DNA (SEQ ID NO: 24)ATGGCGCGCC TGCCGGTGGC AGCGCAGCCC AAGGAGGCGGCCGTCCAGAG CGGCGCCGGC GACTACCTGC TGGGCATCAAGCGGCTGCGG CGGCTCTACT GCAACGTGGG CATCGGCTTCCACCTCCAGG CGCTCCCCGA CGGCCGCATC GGCGGCGCGCACGCGGACAC CCGCGACAGC CTGCTGGAGC TCTCGCCCGTGGAGCGGGGC GTGGTGAGCA TCTTCGGCGT GGCCAGCCGGTTCTTCGTGG CCATGAGCAG CAAGGGCAAG CTCTATGGCTCGCCCTTCTT CACCGATGAG TGCACGTTCA AGGAGATTCT CCTTCCCAAC  GGNTACAACG CCTACGAGTC CTACAAGTACCCCGGCATGT TCATCGCCCT GAGCAAGAAT GGGAAGACCAAGAAGGGGAA CCGAGTGTCG CCCACCATGA AGGTCACCCA CTTCCTCCCC AGGCTGwherein N is selected from A, C, G or T.Sequence of R64M-FGF9 DNA disclosed in PCT application WO 02/36732(SEQ ID NO: 25) A TGCAGCTATA CTGCAGGACT GGATTTCACT TAGAAATCTTCCCCAATGGT ACTATCCAGG GAACCAGGAA AGACCACAGCCGATTTGGCA TTCTGGAATT TATCAGTATA GCAGTGGGCCTGGTCAGCAT TCGAGGCGTG GACAGTGGAC TCTACCTCGGGATGAATGAG AAGGGGGAGC TGTATGGATC AGAAAAACTAACCCAAGAGT GTGTATTCAG AGAACAGTTC GAAGAAAACTGGTATAATAC GTACTCGTCA AACCTATATA AGCACGTGGACACTGGAAGG CGATACTATG TTGCATTAAA TAAAGATGGGACCCCGAGAG AAGGGACTAG GACTAAACGG CACCAGAAATTCACACATTT TTTACCTAGA CCAGTGGACC CCGACAAAGTACCTGAACTG TATAAGGATA TTCTAAGCCA AAGTTGA Sequence of R64M-FGF9-W144X DNA(SEQ ID NO: 26) A TGCAGCTATA CTGCAGGACT GGATTTCACT TAGAAATCTTCCCCAATGGT ACTATCCAGG GAACCAGGAA AGACCACAGCCGATTTGGCA TTCTGGAATT TATCAGTATA GCAGTGGGCCTGGTCAGCAT TCGAGGCGTG GACAGTGGAC TCTACCTCGGGATGAATGAG AAGGGGGAGC TGTATGGATC AGAAAAACTAACCCAAGAGT GTGTATTCAG AGAACAGTTC GAAGAAAAC N NNTATAATAC GTACTCGTCA AACCTATATA AGCACGTGGACACTGGAAGG CGATACTATG TTGCATTAAA TAAAGATGGGACCCCGAGAG AAGGGACTAG GACTAAACGG CACCAGAAATTCACACATTT TTTACCTAGA CCAGTGGACC CCGACAAAGTACCTGAACTG TATAAGGATA TTCTAAGCCA AAGTTGAwherein NNN is other than a codon coding forTrp (TGG) or a stop codon (TAA, TAG or TGA) andis more preferably a codon for amino acid Gly, Arg, Val or Glu.

Sequence of FGF9-2 DNA disclosed in PCT application WO 02/36732(SEQ ID NO: 27) A TGCAGCTATA CTGCAGGACT GGATTTCACT TAGAAATCTTCCCCAATGGT ACTATCCAGG GAACCAGGAA AGACCACAGCCGATTTGGCA TTCTGGAATT TATCAGTATA GCAGTGGGCCTGGTCAGCAT TCGAGGCGTG GACAGTGGAC TCTACCTCGGGATGAATGAG AAGGGGGAGC TGTATGGATC AGAAAAACTAACCCAAGAGT GTGTATTCAG AGAACAGTTC GAAGAAAACTGGTATAATAC GTACTCGTCA AACCTATATA AGCACGTGGACACTGGAAGG CGATACTATG TTGCATTAAA TAAAGATGGGACCCCGAGAG AAGGGACTAG GACTAAACGG CACCAGAAAT TCACACATTT TTTACCTAGA TGASequence of FGF9-2-W144X DNA (SEQ ID NO: 28) ATGCAGCTATA CTGCAGGACT GGATTTCACT TAGAAATCTTCCCCAATGGT ACTATCCAGG GAACCAGGAA AGACCACAGCCGATTTGGCA TTCTGGAATT TATCAGTATA GCAGTGGGCCTGGTCAGCAT TCGAGGCGTG GACAGTGGAC TCTACCTCGGGATGAATGAG AAGGGGGAGC TGTATGGATC AGAAAAACTAACCCAAGAGT GTGTATTCAG AGAACAGTTC GAAGAAAACNNNTATAATAC GTACTCGTCA AACCTATATA AGCACGTGGACACTGGAAGG CGATACTATG TTGCATTAAA TAAAGATGGGACCCCGAGAG AAGGGACTAG GACTAAACGG CACCAGAAAT TCACACATTT TTTACCTAGA TGAwherein NNN is other than a codon coding for Trp(TGG) or a stop codon (TAA, TAG or TGA) and ismore preferably a codon for amino acid Gly, Arg, Val or Glu.

Sequence of R64M-FGF9-N143X DNA (SEQ ID NO: 29) ATGCAGCTATA CTGCAGGACT GGATTTCACT TAGAAATCTTCCCCAATGGT ACTATCCAGG GAACCAGGAA AGACCACAGCCGATTTGGCA TTCTGGAATT TATCAGTATA GCAGTGGGCCTGGTCAGCAT TCGAGGCGTG GACAGTGGAC TCTACCTCGGGATGAATGAG AAGGGGGAGC TGTATGGATC AGAAAAACTAACCCAAGAGT GTGTATTCAG AGAACAGTTC GAAGAA NNN TGGTATAATAC GTACTCGTCA AACCTATATA AGCACGTGGACACTGGAAGG CGATACTATG TTGCATTAAA TAAAGATGGGACCCCGAGAG AAGGGACTAG GACTAAACGG CACCAGAAATTCACACATTT TTTACCTAGA CCAGTGGACC CCGACAAAGTACCTGAACTG TATAAGGATA TTCTAAGCCA AAGTTGAwherein NNN is other than a codon coding for Trp(AAT, AAC) or a stop codon (TAA, TAG or TGA) andis more preferably a codon for amino acid Ser.

Sequence of FGF9-2-N143X DNA (SEQ ID NO: 30) ATGCAGCTATA CTGCAGGACT GGATTTCACT TAGAAATCTTCCCCAATGGT ACTATCCAGG GAACCAGGAA AGACCACAGCCGATTTGGCA TTCTGGAATT TATCAGTATA GCAGTGGGCCTGGTCAGCAT TCGAGGCGTG GACAGTGGAC TCTACCTCGGGATGAATGAG AAGGGGGAGC TGTATGGATC AGAAAAACTAACCCAAGAGT GTGTATTCAG AGAACAGTTC GAAGAA NNN TGGTATAATAC GTACTCGTCA AACCTATATA AGCACGTGGACACTGGAAGG CGATACTATG TTGCATTAAA TAAAGATGGGACCCCGAGAG AAGGGACTAG GACTAAACGG CACCAGAAAT TCACACATTT TTTACCTAGA TGAwherein NNN is other than a codon coding forAsn (AAT, AAC) or a stop codon (TAA, TAG or TGA)and is more preferably a codon for amino acid Ser.

Sequence of FGF9-2-W144X-CNP(1-22) DNA (SEQ ID NO: 31) ATGCAGCTATA CTGCAGGACT GGATTTCACT TAGAAATCTTCCCCAATGGT ACTATCCAGG GAACCAGGAA AGACCACAGCCGATTTGGCA TTCTGGAATT TATCAGTATA GCAGTGGGCCTGGTCAGCAT TCGAGGCGTG GACAGTGGAC TCTACCTCGGGATGAATGAG AAGGGGGAGC TGTATGGATC AGAAAAACTAACCCAAGAGT GTGTATTCAG AGAACAGTTC GAAGAAAAC N NNTATAATAC GTACTCGTCA AACCTATATA AGCACGTGGACACTGGAAGG CGATACTATG TTGCATTAAA TAAAGATGGGACCCCGAGAG AAGGGACTAG GACTAAACGG CACCAGAAATTCACACATTT TTTACCTAGA GGAGGGGGAG GTCTGTCCAAAGGTTGCTTC GGCCTCAAGC TGGACCGAAT CGGCTCCATG AGCGGCCTGG GATGTwherein NNN is other than a codon coding forTrp (TGG) or a stop codon (TAA, TAG or TGA)and is more preferably a codon for amino acid Gly, Arg, Val or Glu.

Sequence of CNP(1-22-FGF9-2-W144X DNA (SEQ ID NO: 32)ATGGGTCTGT CCAAAGGTTG CTTCGGCCTC AAGCTGGACCGAATCGGCTC CATGAGCGGC CTGGGATGCG GAGGGGGAGGGGGAGGGGGA GGGCAGCTAT ACTGCAGGAC TGGATTTCACTTAGAAATCT TCCCCAATGG TACTATCCAG GGAACCAGGAAAGACCACAG CCGATTTGGC ATTCTGGAAT TTATCAGTATAGCAGTGGGC CTGGTCAGCA TTCGAGGCGT GGACAGTGGACTCTACCTCG GGATGAATGA GAAGGGGGAG CTGTATGGATCAGAAAAACT AACCCAAGAG TGTGTATTCA GAGAACAGTT CGAAGAAAAC  NNNTATAATA CGTACTCGTC AAACCTATATAAGCACGTGG ACACTGGAAG GCGATACTAT GTTGCATTAAATAAAGATGG GACCCCGAGA GAAGGGACTA GGACTAAACGGCACCAGAAA TTCACACATT TTTTACCTAG Awherein NNN is other than a codon coding forTrp (TGG) or a stop codon (TAA, TAG or TGA)and is more preferably a codon for amino acid Gly, Arg, Val or Glu.

The principles of the invention are demonstrated by means of thefollowing non-limitative examples.

Example 1 Expression of FGF Variants Using High Expression System

Construction of the p89Bluescript (p89BS) Construct

Construction of p89BS was performed as described in copending WO02/022779. The genes encoding the proteins of the present invention wereligated into the NdeI-BamHI digest of the p89BS construct andtransformed into E. coli cells, such as JM109, TG1, TG2, DHa, andXL1blue.

Construction of FGF Variants

Construction of the FGF-2, FGF-4 and FGF-9 variants was performed usingthe polymerase chain reaction (PCR) technique. Three constitutive PCRreactions were performed, where the variation or variations wereintroduced into the gene by amplifying DNA fragments from both ends ofthe mutation site(s).

The primers and protocol used for the human FGF-2 variants were asfollows:

HF2-for (SEQ ID NO: 58) 5′ GGAATTCCATATGGCTGAAGGGGAAATC HF2-rev(SEQ ID NO: 59) 5′ CGGGATCCTCAGCTCTTAGCAG N111G-for (SEQ ID NO: 60) 5′GATTGGAATCTAATGGCTACAATACTTAC N111G-rev (SEQ ID NO: 61) 5′GTAAGTATTGTAGCCATTAGATTCCAATC N111R-for (SEQ ID NO: 62) 5′GATTGGAATCTAATCGCTACAATACTTAC N111R-rev (SEQ ID NO: 63) 5′GTAAGTATTGTAGCGATTAGATTCCAATC 3, 5Q-for (SEQ ID NO: 64) 5′GGAATTCCATATGGCTCAAGGGCAAATCACCACGCTG(CATATG—NdeI, GGATCC—BamHI, GAATTC-EcoRI restriction sites for cloning)

1. PCR for 5′ domain: The following primers were used: HF2-for or3Q5Q-for and N111G/R-rev

-   -   PCR for 3′ domain: The following primers were used: N111G/R-for        and HF2-rev on the template of human FGF-2 (hFGF2) cloned into        the p80Bs vector.

2. For the entire gene the following primers were used: HF2-for or3,5Q-for and HF2-rev on the DNA from the 5′ and 3′ domains, above.

The primers and protocol, as disclosed in PCT patent application WO02/36732, used to prepare the human FGF-9 variants were as follows:

W144G-for (SEQ ID NO: 65) 5′-CGAAGAAAACGGGTATAATACGTAC W144G-back(SEQ ID NO: 66) 5′-GTACGTATTATACCCGTTTTCTTCG W144R-for (SEQ ID NO: 67)5′-CGAAGAAAACCGGTATAATACG W144R-back (SEQ ID NO: 68)5′-CGTATTATACCGGTTTTCTTCG W144V-for (SEQ ID NO: 69)5′-CGAAGAAAACGTGTATAATACG W144V-back (SEQ ID NO: 70)5′-CGTATTATACACGTTTTCTTCG W144E-for (SEQ ID NO: 71)5′-CGAAGAAAACGAGTATAATACG W144E-back (SEQ ID NO: 72)5′-CGTATTATACTCGTTTTCTTCG W144A-for (SEQ ID NO: 73)5′-CGAAGAAAACGCGTATAATACG W144A-back (SEQ ID NO: 74)5′-CGTATTATACGCGTTTTCTTCG W144N-for (SEQ ID NO: 75)5′-CGAAGAAAACAATTATAATACG W144N-back (SEQ ID NO: 76)5′-CGTATTATAATTGTTTTCTTCG FGF9-Stopback (SEQ ID NO: 77) 5′AGCTGGATCCTCAACTTTGGCTTAGAATATCC R64M-FGF9-for (SEQ ID NO: 78) 5′GGGAATTCCATATGCAGCTATACTGCAGGACTG N143S-for (SEQ ID NO: 79)5′-GTTCGAAGAAAGCTGGTATAATATACG N143S-back (SEQ ID NO: 80)5′-CGTATTATACCAGCTTTCTTCGAACFor example:W144G-for codes for the 5′ to 3′ sequence of the mutation Trp144 intoGly in FGF-9.W144G-back codes for the 3′ to 5′ sequence of the mutation Trp144 intoGly in FGF-9.W144R-for codes for the 5′ to 3′ sequence of the mutation Trp144 intoArg in FGF-9.W144R-back codes for the 3′ to 5′ sequence of the mutation Trp144 intoArg in FGF-9.N143S-for codes for the 5′ to 3′ sequence of the mutation Asn143 intoSer in FGF-9.N143S-for codes for the 3′ to 5′ sequence of the mutation Asn143 intoSer in FGF-9.

The PCR conditions were as follows: annealing temperature was 54° C.followed by elongation at 72° C. for 30 cycles. The purified PCRfragment was digested with NdeI and BamHI, and ligated into the p89BSvector.

FGF-4 Variant

To synthesize the human FGF-4 variants, FGF4-N165G and L55M-FGF4-N165Gcombinations of the following PCR primers were used:

L55M-hF4-for (SEQ ID NO: 81) 5′ ACGTCATATGTTGGCGCGCCTGCCGGTG hF4-rev(SEQ ID NO: 82) 5′ ACGTGGATCCTCACAGCCTGGGGAGGAAG N165R-for(SEQ ID NO: 83) 5′ GATTCTCCTTCCCAACAGGTACAACGCCTACGAG N165R-rev(SEQ ID NO: 84) 5′ CTCGTAGGCGTTGTACCTGTTGGGAAGGAGAATC

L55M-hF4-for and N165G-rev were used to amplify the 5′ domain of humanFGF-4 and incorporate an Met at position 55 and a Gly at position 165.The hF4-rev and N165G-for primers were used to amplify the 3′ domain ofFGF-4 and incorporate the Gly at position 165. The amplified fragmentswere combined and serve as a template for an additional PCR reactionusing L55M-F4-for and hF4-rev. The PCR conditions were as follows: 8cycles with annealing at 50° C., elongation at 72° C. followed by 17cycles with annealing at 60° C., elongation at 72° C. The PCR fragmentwas digested with NdeI and BamHI, gel purified and ligated into thep80BS vector. The bold, underlined bases in SEQ ID NO:81 encode thesubstituted amino acid.

Protein Purification

The newly constructed expression plasmids were transfected intocompetent JM109 bacteria, plated on 2YT-agar plates supplemented with200 ug/ml ampicillin and left to grow ON (overnight) at 37° C. A singlecolony was grown ON at 37° C. in a two-liter flask containing 330 ml ofTB125 medium (Tryptone15 gr/L, Yeast extract 30 gr/L, KH₂PO₄ 2.31 gr/L,K₂HPO₄ 12.5 gr/L, Glycerol 5 gr/L) supplemented with 200 ug/mlampicillin. The bacterial suspension was centrifuged at 4000 rpm (4° C.)for 15 minutes, and the medium was discarded. The bacterial pellet wasthen suspended in 25 ml of 1×PBS buffer containing protease inhibitors,sonicated on ice, and centrifuged at 10,000 rpm (4° C.) for 15 minutes.The protein supernatant was collected, and 3 ml of heparin-Sepharose®beads slurry was added and shaken gently for 6 hours at 4° C. The beadswere loaded onto a column, washed extensively with PBS buffer containing0.3M NaCl, and eluted in 7 ml PBS containing 2-2.5M NaCl. The FGFvariant proteins were then dialyzed against 1×PBS and repurified on FPLCusing a heparin Sepharose® column (HiTrap®Heparin, Amersham Pharmaciabiotech) with a 0-2.5M NaCl (in PBS-0.05% CHAPS) linear gradient in thesame dialysis buffer. The purified proteins were later stored at −70° C.Note that the FGF-9 variants were eluted with 2.5 M NaCl, while theFGF-2 variants were eluted with 2 M NaCl.

Example 2 Preparation of Truncated FGF Variants

The truncated mutants were prepared by PCR, where exemplary primers usedare listed herein below:

35421 (SEQ ID NO: 85) 5′-GGCCCTAGGTCATCTAGGTAAAAAATGTGTG 35422(SEQ ID NO: 86) 5′-GGGAATTCCATATGCAGCTATACTGCAGGACTG 29522(SEQ ID NO: 87) 5′-AGCTGGATCCTCAACTTTGGCTTAGAATATCC 40869(SEQ ID NO: 88) 5′-CGATACGTACATATGCACTTAGAAATCTTC

Where:

35421 was used to introduce stop codon (Pro191Stop) and a BamHIrestriction enzyme site for the construction of the FGF9-2 and FGF9-L72Mvariants;

35422 was used to introduce the start codon and an NdeI restrictionenzyme site for the construction of the R64M-FGF9 and FGF9-2 variants;

29522 was used to introduce the start codon (R64M) and a Bam HIrestriction site for the construction of the R64M-FGF9 variant;

40869 was used to introduce a start codon (L72M) and a BamHI restrictionenzyme site for the construction of the FGF9-L72M variant.

The new mutant PCR fragments synthesized in methods known in the art,were digested with restriction enzymes Nde I and BamHI and cloned inp89BS, forming DNA constructs which were introduced intoelectrocompetent E. coli TG-1 cells.

FIG. 5A depicts the electrophoretic pattern of several of the preferredvariants on SDS-PAGE. Lane 1 contains molecular weight markers [Lysozyme(20.7 kDa), Soybean trypsin inhibitor (28.8 kDa), Carbonic anhydrase(34.3 kDa), Ovalbumin (50 kDa)]; Lane 2 contains native FGF-9; Lane 3contains a 172 aa variant; Lane 4 contains a 164 aa variant; Lane 5contains the R64M-FGF9 (145 aa) variant; Lane 6 contains the FGF9-2variant.

Example 3 FGF Variant Binding to FGFR-Transfected FDCP Cell Lines

The FDCP cell line is a murine immortalized, interleukin 3(IL-3)-dependent cell line of myelocytic bone marrow origin that doesnot express endogenous FGF Receptors (FGFR). Upon transfection with FGFRcDNA, the FDCP cell line exhibits a dose-dependent proliferativeresponse to FGF that can replace the dependence on IL-3. FGFRtransfected FDCP cells can therefore be used to screen variant FGFs forspecific inhibitors, activators or for FGFR signaling. FDCP cellsresponse to various ligands is quantitated by a cell proliferation assaywith XTT reagent (Cell Proliferation Kit, Biological Industries Co.).The method is based on the capability of mitochondrial enzymes to reducetetrazolium salts into a colorogenic compound, which can be quantitatedand is indicative of cell viability.

Specifically, FDCP cells stabily expressing FGFR3-IIIc, FGFR3-IIIbisoforms, FGFR2IIIc or FGFR1 were grown in “full medium” (Iscove'sMedium containing 2 ml glutamine, 10% FCS, 100 ug/ml penicillin, 100ug/ml streptomycin) supplemented with 5 ug/ml heparin and 10 ng/ml FGF.Cells were split every 3 days and kept in culture for up to one month.One day prior to the experiment the cells were split. Before theexperiment the cells were washed 3 times (1000 rpm, 6 min) with fullmedium. The cells were resuspended and counted with Trypan Blue. Twentythousand (2×10⁴) cells were added to each well of 96-well plate in 50 ulfull medium containing heparin. Conditioned medium containing FGF wildtype parent or variants at varying concentrations with heparin was addedin an additional volume of 50 ul full medium to bring the final volumeto 100 ul. The plate was incubated for 48 hours at 37° C. To assay cellproliferation, 100 ul of PMS reagent was added to 5 ml of XTT reagentand mixed well (according to manufacturer's protocol). 50 ul of thelatter solution were aliquoted into each well, and the plates incubatedat 37° C. for 4 hours and the color developed was read by aspectro-ELISA reader at A_(490nm).

In these experiments FDCP cells expressing the FGFR3 isoforms FGFR#IIIband FGFR3IIIc, FGFR2 or FGFR1 were grown in the presence of varyingconcentrations of the FGF-2 and FGF-4 variants.

Results

FIGS. 1A, 1B, 2A and 3 depict the mitogenicity level and receptorspecificity of a sample of the variants of the invention. Wild type(native, parent) FGF-2 or FGF-4 are present as control in the assays.

FIGS. 1A and 1B shows the increase in mitogenicity afforded by theFGF2-N111R variant on FGFR1 and FGFR2 expressing cells.

FIG. 2A shows the increase in mitogenicity afforded by theFGF2(3,5Q)-N111G (closed shapes) variant on FGFR1 and FGFR3IIIc(expressing) cells. The wild type FGF-2 is represented by open shapes.FIG. 2B shows the activity of the same variant in a differentexpreriment on cells expressing FGFR1, FGFR2, FGFR3IIIb or FGFR3IIIc.From these assays the EC50 (effective concentration) of theFGF2(3,5Q)-N111G variant was calculated. On FGFR1 expressing cells theEC50 ranges from about 0.35 ng/ml to 1.0 ng/ml, and on FGFR3IIIcexpressing cells the EC50 ranges from about 0.3 ng/ml to 0.65 ng/ml. TheEC50 of FGF-2 wildtype protein is approximately 2.65 ng/ml on FGFR1cells and 4.2 ng/ml on FGFR3IIIc cells.

FIG. 3 shows the dependency of the FGF2(3,5Q)-N111G variant on heparinfor the different receptor types. The x-axis represents an increasingconcentration of heparin, the concentration of the FGF2v was constant at10 ng/ml.

FIG. 4 shows the induction of mitogenesis afforded by theL55M-FGF4-N165G variant on FGFR1, FGFR2 and FGFR3IIIc expressing FDCPcells. The wildtype FGF4 is a weak inducer of proliferation on FGFR3cells while the L55M-FGF4-N165G variant has converted into a potentmitogen.

FIG. 5B shows the reduced level of proliferation induced by R64M-FGF9both on FGFR3IIIc and FGFR1 cells as compared to parent FGF-9. Theenhanced receptor specificity for FGFR3 permits the use of this variantto target to this specific receptor.

FIG. 6 shows the results of the R64M-FGF9 and FGF9-2 variants in abinding assay as described in Example 4. R64M-FGF9 and FGF9-2 appear tofunction as antagonists of FGF-9.

Example 4 Binding Assay of Truncated FGF Variants to Soluble FGFReceptor Dimer

Binding of FGF proteins to different FGF receptors are determined bymeasuring the degree of competition for binding to different types ofFGFR proteins between a radioiodinated FGF protein and variousunlabelled proteins, or by the direct binding of radioiodinated FGF's tovarious receptor proteins. Binding studies are confirmed by chemicalcross-linking of the radioiodinated FGF to soluble receptors in thepresence and absence of excess unlabelled FGF.

Sodium heparin from porcine intestinal mucosa (PM-heparin) was obtainedfrom Hepar Industries (Franklin, Ohio). KGF is obtained from UBI (LakePlacid, N.Y.). ¹²⁵I was purchased from Amersham (Buckinghamshire,England). FGFs were iodinated using chloramine T. Saline contains 0.05%trypsin, 0.01M sodium phosphate, and 0.02% EDTA (STV). Tissue culturedishes were from Falcon Labware Division, Becton Dickinson (USA),four-well tissue culture plates from Nunc (Rosklide, Denmark).

Soluble FGF receptor proteins were constructed by cloning of theextracellular region of murine FGF receptor 1 (FGFR-1; flg), FGFreceptor 2 (FGFR-2; bek), or the KGF receptor (FGFR3(IIIb) or FGFR3IIIc;K-sam) receptors into the alkaline phosphatase-tag expression vector,which encodes for a secreted form of placental alkaline phosphatase(AP). The FGF receptor alkaline phosphatase (FRAP) plasmids werecotransfected into NIH 3T3 cells by electroporation with a selectableneomycin resistance gene. Colonies were selected in G418 (600 μg/ml) andscreened for secreted AP enzyme activity in the conditioned medium.Clones which produced a high level of AP activity (2 to 4 A₄₀₅units/min/ml) were then used to produce conditioned medium for bindingassays.

Components of the soluble receptor binding reaction mixture includedFRAP-conditioned medium (0.24 OD units/min), 2 ng/ml 125 I-FGFs and 200ng/ml heparin. The FGF:heparin:FRAP ternary complex isimmunoprecipitated with 20 μl of a 1:1 slurry of anti-AP monoclonalantibodies coupled to protein A Sepharose®. All components were mixed atroom temperature. The total volume was adjusted to 200 μl by addition ofDMEM containing 0.1% bovine serum albumin. Binding was allowed toproceed for 1 to 2 hours at 24° C., after which time bound receptorcomplex or the ligand was recovered by centrifugation at 4° C. (10 s at2,000×g). The pelleted material was washed twice with 500 μl of an icecold buffer containing HEPES (20 mM), NaCl (150 mM), glycerol (10%) andTriton X-100 (1%). ¹²⁵I-FGF binding was quantitated by counting of thesamples in a gamma counter. Alternatively, AP enzyme activity of theFRAP protein is determined by transferring the FRAP receptor bound toheparin-Sepharose® to a flat-bottom microtiter plate in a volume of 50μl of PBS. The reaction is initiated by addition of substrate (50 μl of2× solution of AP assay buffer containing 2M diethanolamine, 1 mM MgCl₂,20 mM homoarginine and 12 mM p-nitrophenyl phosphate). The reaction isfollowed at room temperature at 405 nm in a kinetic microplate reader.

Receptor binding was determined by quantitating release of labeled FGFfrom receptors. Briefly, FGF bound to heparan sulfate low affinity sitesis released from the cell surface by a 5 minute incubation with an icecold solution containing 1.6M NaCl, 20 mM HEPES, pH 7.4, and the amountof radioactivity release determined in a gamma-counter. FGF bound tohigh affinity receptors was dissociated by a 2M NaCl (20 mM acetatebuffer, pH 4.0) extraction, and the released labeled FGF is quantitated.

Chemical cross-linking experiments were carried out at room temperaturein a volume of 20 μl in siliconized 0.5-ml microcentrifuge tubes. Thereaction mixtures contain FGF receptor immobilized to anti-AP monoclonalantibodies coupled to protein A Sepharose® was added to give a finalconcentration of 0.15 mM, and the mixture incubated for an additional 30minutes. The reaction was quenched by addition of 1 ml of 200 mMethanolamine-HCl (pH 8.0) for 30 min. The reaction mixtures were diluted1:1 with 2×SDS-polyacrylamide gel electrophoresis loading buffer andelectrophoresed on an SDS-12% polyacrylamide gel. Cross-linked FGF tothe FGF receptor were detected by autoradiography on Kodak XAR film.

Example 5 Effects of FGF Variants on Femoral Growth

Femoral bone cultures are performed by excising the hind limbs of wildtype mice. The limbs are carefully cleaned from the surrounding tissue(skin and muscles) and the femora exposed. The femora are removed andfurther cleared from tissue remains and ligaments. The femora aremeasured for their initial length, using a binocular with an eyepiecemicrometer ruler. The bones are grown in 1 ml of medium with FGF-9,FGF-9 variants, FGF-9 targeting fusion proteins or conjugates, FGF-2 orFGF-2 variants in a 24 well tissue culture dish. The growing medium isα-MEM supplemented with penicillin (100 units/ml), streptomycin (0.1mg/ml) and Nystatin (12.5 units/ml). In addition, the medium containsBSA (0.2%), β-glycerophosphate (1 mM) and freshly prepared ascorbic acid(50 μg/ml). The bones are cultured for 15 days. Measurements of bonelength and medium replacement are performed every three days.

At the end of the experiment, the growth rate of the bones aredetermined from the slope of a linear regression fit on the lengthmeasurements obtained from day 3 to 12. Units given can be converted tolength, 40 units=1 mm.

Example 6 Effect of FGF-2 Variants in Bone Fracture Healing

Suitable animal models are used to create bilateral osteotomies todemonstrate the efficacy of the novel variants of the present invention.In a rabbit model a 6 mm osteotomy is created in New Zealand Rabbits incompliance with the Animal Care Committee of the Hebrew University. Theulna was chosen because it is only slightly weight-bearing and allowsthe creation of a bone defect without requiring a cast or otherimmobilization treatment. In addition, this gap constitutes aspontaneously healing defect that allows the evaluation of the testedagent. The primary indices of fracture healing are accelerated durationof healing and callus formation. The test compounds consist of FGF-2variants in a polymeric scaffold which facilitates bone growth.

Surgical Procedure:

Animals are anesthetized according to standard protocol. Gap formationis performed in the mid Ulna bone. A standard volume of 0.2 ml oftreatment formulation is put into the gap area in each limb and thefracture is closed. Animals are treated with analgesics for 3 days postoperation. The duration of the experiment is 6 weeks.

Healing Time and Quality Assessment:

Healing time evaluation: X-ray grading provides fracture healing statusassessment. Rabbits are x-rayed every other week for 5 weeks aftersurgery. Two orthopedic surgeons score X-rays in a blinded manneraccording to standard grading scale protocol.

Quality evaluation: at the end of the experiment rabbits are sacrificedand fracture area is sent for histological and mechanical strengthevaluation. Histology is scored by a pathologist for evaluation ofhistological changes during the healing process using standard stainingmethods, using hematoxylin and eosin for cytoplasm and nucleus.Indigo-Carmin staining is also applied for detection of newly generatedcallus. Mechanical strength evaluation is performed using the “4 pointsbending” method.

The treatments groups are: Osteotomy without treatment, Osteotomytreated with polymeric scaffold alone, Osteotomy treated with scaffoldcontaining FGF-2 and an osteotomy treated with scaffold containing FGF-2variant, FGF2(3,5Q)-N111G.

X-Ray Scoring

0—No callus1—Primary callus response at one end of bone2—Primary callus response at both ends of bone3—Partial external callus union4—Complete external callus union5—<30% gap closure6—>30% gap closure7—Complete gap closure8—Partial callus remodeling9—Complete callus remodeling

Gap Filling Calculation:

(A/A+B)×100=% gap filling

Example 7 Efficacy of FGF Variants in Distraction Osteogenesis

Distraction osteogenesis is a useful method for bone elongation ofextremities in short stature and for the treatment of extensive bonedefects. Several procedures for bone lengthening have been developed foruse in the clinic. The problems encountered in using this techniqueinclude an extended healing time and complications such as non-union orpoor quality of the regenerated bone.

The maximal rate of elongation used in the current procedure of limbelongation, while maintaining proper bone healing and reconstitution, is1 mm/day. Faster elongation rates have resulted in lack of fusion or inthe formation of weak bone that breaks easily or cannot bear bodyweight. In this process, extreme conditions of elongation (1.5 mm/day)will be performed in order to observe a more significant effect of theadded compounds on the background of natural healing.

The objectives of the experiment are to assess the quality of boneformation, time of bone formation and safety after elongation using acalcium phosphate (CaP) scaffold embedded with the FGF2 variant.

Treatment Arms:

Control: 5 lambs (5 limbs), no treatment

Treatment 2: 5 lambs (5 limbs): CaP alone

Treatment 3: 5 lambs (5 limbs): CaP with FGF-2 variant

Materials and Methods:

Lambs are assigned randomly into one of the five treatment arms.

Surgical lengthening of the right femur is performed in 25 lambs agedfrom 3 to 4 months.

Anesthesia and Pre-Mediation:

General anesthesia is given without endotracheal intubation.Intramuscular atropine is given as premedication (0.5 mg/kg), andthiopentone sodium-2.5% (10-15 mg/kg), Fentanyl® (0.0015 mg/kg) andDiazepam® (0.2 mg/kg) is administered intravenously.

Fixation:

A monolateral external fixator (Monotube-Triax®, Stryker Trauma, Geneva,Switzerland) with four pins, two proximal and two distal in each of itspin clamps, is positioned so that the pins are kept away from the growthplates and the surface of the joint. The osteotomy is performed using apneumatic saw.

Lengthening:

Lengthening begins seven days after surgery for all treatment groups:

Lengthening continues until the limb has been lengthened by 4.5 cm. Thetotal elongation period lasts 30 days at a rate of 1.5 mm/day startingthe 8^(th) day after surgery.

Treatment:

Lambs are assigned randomly into one of the four treatment arms. Alltreatments take place during the consolidation period, at day 44.

Treatment 1—Control—To assess the effect during the consolidationperiod, animals remain without treatment until the end of the trialperiod.

Treatment 2—To assess the effect of CaP alone during the consolidationperiod, it will be administered once, one week after completion ofelongation.

Treatment 3—To assess the effect of the variant protein duringconsolidation period, CaP with FGF-2 variant, FGF2-N111G orFGF2(3,5Q)-N111G is administered once, one week after completion ofelongation.

Follow Up:

Animals are in a restricted area during the extent of the wholeexperiment and are allowed to feed and walk ad libitum in own cage.Animals are weighed at fixed intervals and general well-being ismonitored.

To study the bone formation in the host bone, four differentfluorochromes are used as bone markers, administered IM, according tothe following schedule: one week after surgery: calcein (green)(Sigma®); two weeks after surgery: alizarin (red) (Sigma®); three weeksafter surgery: xylenol (orange) (Fluka®) and three days before sacrificeoxytetracycline will be given (Duphacycline®). The Spalteholz techniqueis performed after intra-arterial injection of Berlin blue studiedthrough the femoral artery before sacrifice to analyze thevascularization of the lengthened callus in each group.

Completion:

The animals are sacrificed three months after initial surgery by IVinjection of 5 meq of KCl, after anesthesia with sodium pentobarbital(1.5 mg/kg weight).

Assessment of Efficacy:

Success is determined in terms of healing time and bone quality obtainedafter elongation and treatment with FGF2 or FGF2 variant and if no majoradverse effects are observed.

X-Ray

Progress of bone healing is followed by X-ray at weeks 1, 2 and 4 afterbeginning of elongation.

The parameters to be assessed from the X-ray are:

1. Degree of callus formation,

2. Gap closure

3. Remodeling achieved during treatment.

X ray scoring is performed by an orthopedic surgeon, according to anestablished bone healing grading system.

Histology

The callus is divided into two parts, one for embedding in paraffin, andthe other undecalcified, for embedding in methylmethacrylate. For thehistological study, the specimens will be fixed in Bouin for 24 hoursand decalcified in a solution of PVP-EDTA, at 4° C. Once specimens havebeen decalcified, they are dehydrated using increasing concentration ofalcohols (70%, 80%, 96% and 100%), and after 4 hours in xylene, they areembedded in paraffin at a temperature of 60° C. The specimens aresectioned to 4 μm, and stained with Masson's trichrome, hematoxylin andeosin (H&E), safranin 0 and von Kossa.

To analyze the mineralization by fluorochromes, the specimens are fixedin formol for one week, then dehydrated using alcohols of increasingproof. After one week in PMMA-alcohol and three weeks in PMMA (Technovit7200 VLC®), specimens will be sectioned with a diamond saw (Exakt®) andtrimmed to a thickness of 14 μm. After measuring the sections withultraviolet light the distance of the bone markers is measured and thebone index formation calculated (distance mm/days)

The proximal parts of both, lengthened and control, tibiae are extractedand cut in lateral and medial parts. The lateral portion is placed in 4%buffered formaldehyde. After decalcification of all the specimens inEDTA, are proceed to embed them in paraffin and cut them into 4 μmslices. Stains of H&E, Masson's trichrome, Safranin 0 and Alcianblue-PAS are applied.

Immunohistochemistry

Specific antibodies recognizing collagen I, collagen II, FGFa (now knownas FGF-1), and S-100 are applied to the lengthened callus sections by anindirect two-step method. The 4 μm paraffin sections are dewaxed inxylene and taken through ethanol 100%. After trypsinization, followingdeparaffinization, endogenous peroxidase is blocked by placing thesections in hydrogen peroxidase solution for 30 min. They are thenincubated in the following reagents with appropriateTris-buffered-saline (TBS: 0.55 M, pH 7.36) washes: normal pig serum for30 min, abovementioned primary antibodies for 1 hour, a secondarybiotinylated antibody for 30 min, and avidin-biotin complex (Dako KO335)for 30 min. The reaction is visualized with chromogen substrate solution(diaminobenzidine, hydrogen peroxidase, TB) and sections arecounterstained with Harris's hematoxylin, dehydrated, and mounted. As anegative control, TBS is used in the procedure instead of the primaryantibodies. All stained sections are examined and photographed with useof a microscope (Nikon Optiphot-2®, Japan).

Morphometric Analysis

With an image analyzing system (Leica Q 500 MC®) the histomorphometricparameters are determined. With Masson's trichrome stain the followingparameters are determined:

1. Trabecular width;

2. Trabecular area;

3. Trabecular erosion surface;

4. Index of trabecular erosion;

5. Number of osteoblasts;

6. Number of osteoclasts per field;

7. Number of osteoclast nuclei;

8. Index of bone reabsorption or number osteoclast nuclei/osteoclasts.

With von Kossa's stain the following parameters are obtained:

1. Osteoid width;

2. Osteoid—trabecular index, and fluorescence will be used to measure;

3. Bone formation index.

Example 8 Targeting of FGF Variants

The FGF-9 variants having the ability to bind the FGFR3 yet having areduced capacity to effect a biological response can be used astargeting vectors for the different bioactive agents. CNP is known toincrease bone length (see Example 9). CNP derivatives include CNP(1-22),CNP(1-17) and derivatives thereof wherein stability or half life isincreased. In particular, FGF9-2-W144G, a 127 amino acid variant whichcomprises both N- and C-termini truncations and an amino acidsubstitution at tryptophan 144 (W144), was shown to target efficientlyto the growth plate of long bones.

One day old mice pups were injected IP with iodinated FGF9-2-W144G.Animals were sacrificed 2 and 8 hrs later and whole embryo sections wereperformed. In these pups, the labeled FGF was observed to localize tothe growth plate of the hind limb, close to the site of injection. FIGS.7A and B shows two exposures of the distribution of I¹²⁵ FGF9-2-W144G inthe mouse growth plate following IP delivery. P, M, H and T define theproliferating, maturating, hypertrophic and trabecular regions,respectively, of the growth plate. FIG. 7B shows the outline of thecells. FIG. 7A shows strong staining in the hypertrophic zone and somesignal in the proliferative and trabecular regions. No other specificsites were labeled by this FGF9 variant. This experiment shows deliveryof an exogenously administered compound to the growth plate in vivo andprovides a tool for targeted delivery of factors such as natriuretcpeptides (NP) or NP analogs. The production of the fusion constructs isillustrated in Example 9. During fetal life and until the end ofpuberty, longitudinal bone growth takes place via endochondralossification of the growth plate located at the epiphyses (ends) of longbones. The growth plate is divided into several zones of cartilageforming cells, or chondrocytes, with distinct patterns of geneexpression. In the Reserve Zone, cells are small and relativelyinactive. In the adjacent Proliferative Zone, chondrocytes proliferate,arrange themselves in columns and eventually undergo hypertrophy. In theLower Hypertrophic Region towards the cartilage-bone junction, cells arebig and highly active but exhibit no further cell division. The matrixsurrounding the hypertrophic cells calcifies and the lowermost cellsundergo programmed cell death. Cell death is accompanied by the removalof the cartilaginous matrix and its replacement by bone through theconcerted action of recruited bone cells, namely osteoclasts andosteoblasts.

Example 9 FGF Variant Fusion Constructs

In addition to members of the FGF family, Natriuretic peptides (NP), andC-type natriuretic peptide (CNP) in particular, have been shown toregulate bone growth. It has been shown that CNP knockout mice whichexhibit skeletal phenotypes histologically similar to those seen inachondroplasia mice (Chusho et al., PNAS 98, 4016, 2001). They alsoreveal the rescue of the CNP knock out skeletal defects bytissue-specific ectopic CNP expression in the growth plate. Moreover, exvivo experiments (fetal bone organ culture) from wild type animals haveshown that CNP, more than BNP and ANP, can induce bone elongation(Yasoda et al., 1998; Mericq et al., 2000). In a currently preferredembodiment of the present invention provided is a method to increase thesize of a bone growth plate by treating the bone with a pharmaceuticalcomposition comprising an FGF variant-NP fusion protein. In a currentlymore preferred embodiment the FGF variant is FGF9-2 and the NP is CNP oran analog thereof.

Two FGF9-2-CNP fusion constructs were prepared, each using fouroligonucleotide primers and three PCR reactions. The first,FGF9-2-W144G-CNP(1-22), wherein FGF9-2 lies in a 5′ orientation to CNP,was constructed as follows:

FGF9-2-for (SEQ ID NO: 89)  5′-GGGAATTCCATATGCAGCTATACTGCAGGACTG CNP-rev(SEQ ID NO: 90) 5′-AGCTGGATCCTCAGCAACCCAGACCGGACATG F9-2-CNP-for(SEQ ID NO: 91) 5′-CACACATTTTTTACCTAGAGGAGGGGGAGGTCTGTCCAAAGGTTGCF9-2-CNP-rev (SEQ ID NO: 92)5′-GCAACCTTTGGACAGACCTCCCCCTCCTCTAGGTAAAAAATGTGTG

The first PCR reaction (20 cycles) was performed using FGF9-2 for andCNP-rev on an FGF9-2 template. The second (20 cycles) was performedusing F9.2-CNP-for and F9-2-CNP-rev on a mCNP (mouse) template. Thethird PCR reaction (20 cycles) was performed using the products of thetwo previous PCR reactions as template and amplifying using FGF9-2-forand F922-CNP-rev.

The second fusion construct, CNP-FGF9-2-W144G, comprises the CNPN-terminal to the FGF9-2. The following primers were used:

CNP-for (SEQ ID NO: 93) 5′-ACGTGACCATATGGGTCTGTCCAAAGGTTG CNP-F9-2-rev(SEQ ID NO: 94) 5′CAGTCCTGCAGTATAGCTGCCCTCCCCCTCCCCCTCCCCCTCCGCAACCCAGACCGGACATGCNP-F9-2-for (SEQ ID NO: 95)5′-ATGTCCGGTCTGGGTTGCGGAGGGGGAGGGGGAGGGGGAGGGCAGCTATACTGCAGGACTGP191Stop (SEQ ID NO: 96) 5′-GGCCCTAGGTCATCTAGGTAAAAAATGTGTG

The first PCR reaction (20 cycles) was performed using CNP-for andCNP-9-2-rev on a mCNP template. The second (20 cycles) was performedusing CNP—F9-2-for and P191Stop on a mCNP template. The third PCRreaction (20 cycles) was performed using the products of the twoprevious PCR reactions as template and amplifying using CNP-for andP191Stop primers.

The PCR products were cloned in an expression vector, p80 Bluescript,sequenced and analyzed for accuracy and used to transfect host cells.Fusion protein was produced by methods known in the art. Fusion proteinsare analyzed for CNP activity using the Biotrak enzyme immunoassay (EIA,Amersham) that measures the amount of secondary messenger, cyclic GMP(cGMP), elicited after activation of the natriuretic peptide receptor bythe peptide on C3H10T1/2 cells.

It will be appreciated by the skilled artisan that the fusion constructcan comprise an FGF variant of the present invention fused to abioactive agent including a peptide or peptide analog or hormone,including growth hormone (GH), IGF-1, TH or PTHrP that istherapeutically beneficial to target to the growth plate.

Example 10 Articular Chondrocyte Culture

Chondrocytes were isolated from pig or human biopsies and cultured inthe presence of the variants of the present invention to identify theeffect of the variants on proliferation and differentiation. Theprocedure employed for the isolation and propagation of articularchondrocytes is presented below.

Reagents: Dulbecco's MEM (DMEM) (Gibco BRL, cat. no. 41965) MEMNon-Essential Amino Acids (Gibco BRL, cat. no. 11140) Sodium Pyruvate(Gibco BRL, cat. no. 11360) Fetal Bovine Serum (FBS) (Gibco BRL, cat.no. 10270)

Streptomycin, Penicillin, Nystatin Solution (Biological Indus. Ltd.,cat. no. 03 0321)Trypsin-EDTA (Gibco BRL, cat. no. T8154) or Versene-Trypsin (Bio LABLtd., cat. no. 13.012)Collagenase Type 2 (Worthington Biochem. Corp. Cat. No. 4147). A stocksolution of 1700 units/ml Collagenase in DMEM was prepared and filtered(0.2 μm).

Preparation of FBS-DMEM Medium:

FBS (50 ml), 5 ml of antibiotic solution, 5 ml Sodium Pyruvate, 5 ml MEMnon-essential amino acids were added to a 500 ml bottle of DMEM. Wherespecified, FGF-2, FGF-9 or FGF variants were added to a finalconcentration of 10 ng/ml.

Isolation of Cells from Cartilage Biopsy:

A piece of cartilage tissue was minced into 2 to 4 mm pieces with asterile scalpel. The collagenase solution was diluted 1:4 in FBS-DMEM,added to the tissue sample and left to incubate on a rotator at 37° C.,overnight (ON). The cells were centrifuged (1200 rpm 5-10 min). Themedium was aspirated, the cells washed in 5 ml medium and recentrifuged.The cells were resuspended in culture medium and seeded in 25 cm² or 75cm² flasks at a concentration of approximately 1×10⁶ cells per flask.The cells were incubated in a 5% CO₂ incubator at 37° C. The cell mediumwas replaced every 2-3 days.

Procedure for Passaging Cells (Trypsinization):

When the cell culture reached the desired confluency the medium wasremoved and the cells trypsinized according to standard procedure. Thecells were split to 2-3 new flasks and 20 ml fresh pre-warmed medium wasadded. The expansion of cells and trypsinization was performed asnecessary.

Furthermore, the cell population grown on the above matrices expressesseveral of the chondrocyte differentiation markers. One of severalphenotypes expressed during chondrocyte differentiation isglycosaminoglycan (GAG) production. The production of GAGs is identifiedin histological staining using Alcian blue or toluidine blue andquantitated using the DMB (3,3′-dimethoxybenzidine dihydrochloride) dyemethod.

Example 11 Cell Proliferation/Differentiation Assay

Articular chondrocytes that have been isolated by enzymatic digestionand maintained in monolayer culture undergo dedifferentiation over timeand shift to a fibroblast-like phenotype. This is reflected in part bytheir morphology and loss of expression of collagen II. The cells areable to undergo proliferation and differentiation into articularchondrocytes under certain growth conditions.

Proliferation of the cartilage cells in the presence of the differentvariants was quantitated by one of two methods, CyQUANT® (MolecularProbes) or XTT reagent (Biological Industries, Co.). Human or porcinearticular chondrocytes (10⁴-10⁵ cells/100 ul) were grown in the presenceof the variants of the invention (10 ng/ml) in microwell plates. Thecells were grown for the several days in DMEM with and without FGF andvariants, and the cells processed according to manufacturersinstructions. The plates were read in an ELISA reader at A490 nm.Results for human articular chondrocytes are shown in FIG. 9A-9E.

Articular chondrocytes were isolated from cartilage tissue fragments.Cells were grown using culture media supplemented with Fetal Calf Serum(FCS). Different concentrations of FGF-2 or FGF-9 or FGF variantsFGF2-N111G, FGF2-N111R or FGF9-W144G were added to the medium and thento the cells. Medium with variant was exchanged every 2-3 days.Proliferation of cells was determined using CyQUANT™ Cell ProliferationAssay Kit (Molecular Probes).

Morphology of Cultured Cells

Human or porcine articular chondrocytes were grown in culture with orwithout wild type and variant FGFs for two weeks. The cells wereobserved under an inverted microscope and stained withfluorescent-conjugated phalloidin.

The data from the human articular chondrocytes are shown in FIGS. 8 and9. FIG. 8 shows the proliferation curve of cells that were cultured inmedium with and without added FGF ligand. The ligands FGF-2, FGF2-N111G(FGFv) and FGF9 have a proliferative effect on the articularchondrocytes, while FGF9-W144G (FGFv) does not enhance proliferation.

FIGS. 9A-9E shows human articular chondrocytes following 2 weeks inculture as seen under an inverted microscope. Cells grown without ligand(panel A) exhibit a fibroblastic morphology with undefined borders,while cells grown in the different ligands have variable polygonalshapes. Without wishing to be bound by theory these results suggest thatthe cells retain the chondrocytic phenotype are able to undergodifferentiation once the cells have been induced to proliferate.Furthermore the cells' volume is affected and the cells grown inFGF-9-W144G (panel E) are the largest. The cells in panel B and C weregrown in FGF-2 or FGF2(3,5Q)-N111R, respectively.

FIG. 10A shows the morphology of porcine articular chondrocytes grownwithout added ligand. The cells are fibroblast-like and have undefinedborders. The cells grown with human FGF2-N111G variant (FIG. 11B) arerounded with highly defined borders, while the cells grown in humanFGF9-W114 variant are cuboidal and resemble articular chondrocytes. Thecells were stained with fluorescent labeled phalloidin to labels theactin cytoskeleton of the cells. The data are presented in FIGS.11A-11C. The differences in the actin cytoskeleton between thetreatments are very clear. The actin of the cells grown in mediumwithout ligand (FIG. 11A) is elongated and typical of fibroblast-likecells. The actin of cells grown in the FGF2-N111R ligand is round anddefined while the actin of cells grown in FGF9-W144G is polygonal.

It is thus possible to alter the cellular phenotype of certain types ofcells by exposing them to FGF variants having at least one amino acidsubstitution in the beta8-beta9 loop.

Example 12 Chondrocyte Pellet Culture

Cell differentiation and morphogenesis was studied in pellet culturesand analyzed by using cell-type-specific markers. 2.5×10⁵ porcinearticular chondrocytes that had been expanded in culture in the with andwithout FGF variants were pelleted in 0.5 ml differentiation medium(DMEM—high glucose containing the following: 1 μM dexamethasone, 1 mMSodium pyruvate, 50-100 ug/ml ascorbic acid, 0.35 mM proline, 10 ng/mlIGF-1, 10 ng/ml TGFβ, Insulin-Transferrin-Selenium solution (6.25 μg/mleach)) and incubated in differentiation medium in 15 ml polypropylenecentrifuge tubes with caps loosened. Medium was exchanged every 2-3days. The pellets were sectioned using standard methods known in the artand stained with toluidine blue to label the sulfated proteoglycans andimmunohistochemically stained with anti-collagen II antibodies.

The pellet culture grown in medium with no ligands or with FGF2-N111Gshowed low collagen II expression. The cultures grown in medium withFGF9-W144G show the appearance of collagen II over time. FIG. 12A showsa small amount of collagen staining after a week, while FIGS. 12B and12C show a high amount of collagen II protein expression after 2 and 6weeks, respectively. FIGS. 13A and 13B show the high level ofproteoglycan staining in the tissue resulting from the cells cultured inmedium comprising the FGF9-W144G variant, while FIGS. 13C and 13D showno toluidine blue staining for cultures grown in medium alone or withFGF-2 variant, respectively.

This result shows that the FGF9-W144G variant is effective indifferentiation of cultured articular chondrocyte cells.

The same experiment is performed on chondrocytes isolated from human andother mammalian sources.

Example 13 Goat Articular Cartilage Repair Model

A comparative study to evaluate the efficacy of the FGF variants intreating articular cartilage defects in a goat knee injury model isperformed. A total of 6 adult female goats are used. All of the animalsundergo a chondrocyte harvest procedure prior to implantation. Thecollected tissue will be used for preparation of autologous primarychondrocytes. Three weeks post operative, a 4.5 mm diameter and 1.5 mmdeep hole are punched out and natural matrix matrices, with or withoutFGF variants, pre-seeded with different concentrations of allogeneiccells are implanted in the corresponding individual goat for a long termexperiment (12 weeks). After 12 weeks, all animals are humanelyeuthanized. The joints are grossly evaluated for specific changes of thefemoral condyle and the contacting surfaces. Histological analysis isperformed to determine the structural and cellular response to theimplant materials.

Materials and Methods:

Six adult female goats (11-12 months old) are used. In one particularexperimental system the different tests include:

Treatment Goat Proximal Treatment Middle Treatment Distal 1-3 MatrixMatrix + 0.4 × 10⁵ cells Matrix + 2 × 10⁵ cells 4-6 Matrix + FGFvMatrix + FGFv + 0.4 × Matrix + FGFv + 2 × 10⁵ cells 10⁵ cells

The variant tested is FGF2(3,5Q)-N111G.

Antibiotics: 2 ml of amoxycillin is injected IM immediately before theprocedure and once a day for 4 days after the procedure.

Anesthesia: Pre-medication: 0.05 mg/kg xylazine followed byketamine-diazepam (4 mg/kg and 2 mg/kg IV) is administered IM.

Surgery and Implantation:

The basic surgical procedure is identical for all subjects. Allsurgeries are performed under strict asepsis. Peri-operative antibioticsare dosed IM at 2.4 million units of Penicillin procaine (40,000units/kg SID) at the beginning of the procedure. Anesthesia is inducedwith xylazine 0.05 mg/kg IM followed by ketamine-diazepam (4 mg/kg and 2mg/kg IV). The subject is intubated in ventral position and thenpositioned to left recumbency. Anesthesia is maintained with a gaseousmixture of Isoflurane and oxygen. Analgesia, carprofen 2-4 mg/kg SQ,SID.

Harvest Procedure:

The surgical approach consists of a curved, lateral skin incision madefrom the distal one-third of the left femur to the level of the tibialplateau and across to the medial side of the tibial spine. Using thismethod, the skin is bluntly dissected and retracted to allow a lateralparapatellar approach into the stifle joint. An incision is madeparallel to the lateral border of the patella and patellar ligament.This extends from the lateral side of the fascia lata along the cranialborder of the biceps femoris and into the lateral fascia of the stiflejoint. The biceps femoris and attached lateral fascia are retractedallowing an incision into the joint capsule. The joint is extended andthe patella luxated medially exposing the stifle joint.

The harvest site is the same as the location of the planned trochleardefect which is created in the right femoral condyle. The defects isapproximately 5 mm in diameter and approximately 2.5 mm in depth, andwill pass into the subchondral bone. The defects are made on either thelateral or medial wall of the distal trochlear sulcus dependent onindividual anatomy. The harvested cartilage layer is approximately 5 mmin diameter and approximately 0.5 mm in depth. The harvested cells aretransferred to cell culture medium immediately after harvest forexpansion and matrix seeding. The incision is closed in layers usingappropriate suture and patterns.

Implantation Procedure:

The trochlear defect is created in the right femoral condyle. Thedefects are approximately 5 mm in diameter and approximately 2.5 mm indepth, and pass into the subchondral bone. The defects are made oneither the lateral or medial wall of the distal trochlear sulcusdependent on individual anatomy. Each defect is filled with theappropriate test article.

The surgical approach consists of a curved, lateral skin incision madefrom the distal one-third of the left femur to the level of the tibialplateau and across to the medial side of the tibial spine. Using thismethod, the skin is bluntly dissected and retracted to allow a lateralparapatellar approach into the stifle joint. An incision is madeparallel to the lateral border of the patella and patellar ligament.This extends from the lateral side of the fascia lata along the cranialborder of the biceps femoris and into the lateral fascia of the stiflejoint. The biceps femoris and attached lateral fascia are retractedallowing exposure to the joint capsule. The joint is extended and thepatella luxated medially exposing the stifle joint.

With the knee joint fully flexed, the appropriate location for thepoints of drilling the defect on the trochlear sulcus are identified andmarked with a surgical marker. A specially designed cartilage cutter isused to slice through the cartilage outer layer and prevent tearing ofthe cartilage. The approximate 5 mm diameter core cutter is used underpower to create a fixed depth of approximately 2.5 mm, maintaining aplane perpendicular to the tangent of the sulcus. The core ofsubchondral bone and cartilage is carefully removed. The cutter iscarefully removed and any loose cartilage edging is carefully dissectedwith a scalpel blade. If needed, a handheld powered drill with aspecially designed drill bit is used to chamfer the edge of the createddefect. This undercutting may assist in providing a mechanical lock withthe matrix.

The cartilage defects are copiously flushed with sterile saline prior toinsertion of the test article. The appropriate test material is thenplaced into the defect such that it is in line with the surroundingcartilage and covered with biological glue to maintain in place. A finalsaline flush of the joint is carefully done. The patella is then reducedand the joint moved through a complete range-of-motion to ensure thatthere is no impingement due to the implants. This is followed by routineclosure of the joint in three or four layers using appropriate suturematerial.

Post operatively, a modified Thomas splint is applied to the leg. Thisremains in place for 2 weeks to limit flexing of the operated knee. Postoperative checks are made for any animal displaying signs of postoperative discomfort. Post operative analgesics are given for 5 days ifthe animals display any signs of distress of discomfort. All treatmentsare recorded in the appropriate study documentation.

Necropsy

Animals are humanely sacrificed at 12 weeks postoperatively. Bodyweightsare recorded immediately prior to sacrifice. Deep anesthesia is inducedwith a mixture of ketamine-xylazine and the subject exsanguinatedaccording to the guidelines set forth by the AVMA Panel on Euthanasia(JAVMA, March 2000).

Gross evaluation and sample collection as described in Table 4 areperformed. Lymph nodes in close proximity to the joint is examined. Thearticulating surfaces opposing the defect sites are examined for anyabnormal joint surface. Additionally, gross evaluations of the kneejoints is made to determine the cartilage repair based on previousscoring criteria listed in Table 4. Femora, patellae, synovium, andpopliteal lymph nodes are harvested and placed into appropriatelylabeled containers. Immediately following tissue harvest, grossmorphological examination of the cartilage surface is done as describedabove and photographic records made of each specimen.

TABLE 4 Gross Evaluation and Sample Collection Gross Sample PhotographSample Evaluation collection and Score Heart Lungs Kidneys SpleenPopliteal lymph nodes X x Knee joint (includes X x x articulating defectsite)

Gross Morphological Observations

After collection of the knee joints, the joints are opened, photographedand the surface of the defect site scored as indicated in Table 5. Thesynovial membrane is examined for any inflammation. Joint fluid iscollected and analyzed.

TABLE 5 Scoring Criteria for Gross Morphological EvaluationsCharacteristic Grading Score Edge Integration Full 2 (new tissuerelative to native cartilage) Partial 1 None 0 Smoothness of thecartilage surface Smooth 2 Intermediate 1 Rough 0 Cartilage surface,degree of filling Flush 2 Slight depression 1 Depressed/overgrown 0Color of cartilage, opacity or Transparent 2 translucency of theneocartilage Translucent 1 Opaque 0

Histology and Histological Evaluation

Immediately after dissection and following gross joint surfaceobservations, the joints is placed in 10% phosphate buffered formalin(at least ten-fold volume) for at least 48 hours and sent forhistological processing. After fixation in 10% phosphate bufferedformalin, the specimens is grossly trimmed to remove extra tissue. Thetissue blocks are cut approximately ⅓ of the distance in from theexterior implant/tissue interface in order to examine them grossly.Contact radiographs is taken prior to the commencement ofdecalcification.

The tissues are decalcified in 10% EDTA until radiographs of thedecalcified sections assures complete decalcification. Once completedecalcification is determined, the specimens is dehydrated through anethanol series and paraffin embedded. The specimens is sectioned to 5-10μm. One section is stained with H&E and another sequential section withSafranin O counter-stained with Fast Green. For histologic analysis ofthe sections, the scoring scale according to Frenkel is used.

Histological evaluation is performed to measure the followingparameters: Characteristics of the neo-formed tissue, regularity of thejoint surface of the regenerated tissue, structural integrity andthickness of the regenerated tissue, endochondral ossification and stateof the cells in the remaining cartilage.

Example 14 Pharmacokinetics

Methods for detecting administered compounds in the blood or tissue oftreated mammals are known in the art. The pharmacokinetic properties ofthe administered compounds are determined using such methods. In animalmodels, radiolabelled oligonucleotides or peptides can be administeredand their distribution within body fluids and tissues assessed byextraction of the oligonucleotides or peptides followed byautoradiography (Agrawal et al PNAS 88,:7595, 1991). Other methodsinclude labeling of a peptide with a reporter moiety, includingfluorescent or enzyme labels, administration to an animal, extraction ofthe peptide from body fluids and organs followed by HPLC analysis.Alternatively, immunohistochemical methods are used for detection of theadministered peptide in tissue. The present invention contemplatesreporter labeled FGF polypeptides and chimeras, fusion protein, hybridsand conjugates using the same.

Example 15 Effect of Variants on PC12 Cells

To investigate whether neuronal PC12 cells respond differently to theFGFs and FGF variants, cells were exposed to FGF-2, FGF2-N111R, FGF-9and FGF9-W144G.

The PC12 cell line was originally cloned from a transplantable ratadrenal medullary pheochromocytoma. The cells were grown in DMEM withhigh glucose supplemented with 10% horse serum, 5% fetal calf serum,130-units/ml penicillin and 0.1 mg/ml streptomycin and 0.25-40 ng/ml FGFor variant in a humidified incubator at 37° C. To harvest the cell, thecell layer was washed with PBS-EDTA. The cells were collected andcentrifuged for 5 min, 2000 rpm. The cells were resuspended in 5 ml DMEMand plated.

After 3 days in culture, both FGF-2 and FGF-9 induced neuronaldifferentiation at a similar level, as determined by the observation ofneurite extensions. Close observation of the cultures demonstrated thatthe length of the neurite outgrowth induced by FGF-2 was typicallylonger than that induced by FGF-9. Importantly, the variants induced aninverse effect when compared to their wild type counterpart. WhileFGF2-N111R was more potent, as determined by the number and length ofthe neurite extensions, than FGF-2, FGF9-W144G had the weakest activityof all tested ligands exerting minimal differentiation even at thehighest concentration employed (40 ng/ml).

The introduction of a mutation in the β8-β9 loop of the FGF-2 and FGF-9ligands resulted in polypeptides having a selective effect on the cellsin culture.

While the present invention has been particularly described, personsskilled in the art will appreciate that many variations andmodifications can be made. Therefore, the invention is not to beconstrued as restricted to the particularly described embodiments,rather the scope, spirit and concept of the invention will be morereadily understood by reference to the claims which follow.

What is claimed is:
 1. A method of treating an individual that (i) hasabnormal bone; or (ii) is afflicted with a disease or disorder relatedto normal or abnormal FGF receptors or a skeletal disorder; or (iii) hasdysplasic bone, which method comprises administering to the individual apharmaceutical composition comprising a therapeutically effective amountof a fibroblast growth factor 9 (FGF-9) variant comprising at least oneamino acid substitution in the beta 8-beta 9 loop, wherein the FGF-9variant is an antagonist; wherein the FGF-9 variant reducesproliferation of target cells having at least one FGF receptor subtypecompared to the corresponding wild type FGF-9; wherein the FGF-9 varianthas enhanced receptor specificity for the at least one receptor subtypecompared to the corresponding wild type FGF-9 by decreasing thebiological activity mediated by at least one receptor subtype whileretaining the activity mediated through another receptor subtype;wherein the FGF-9 variant further comprises a truncation at theN-terminus, or at the C-terminus, or at both termini; and wherein theFGF-9 variant comprises an amino acid sequence selected from one of thesequences forth in SEQ ID NO: 11, 13, 14, 15, 16 and
 17. 2. The methodaccording to claim 1, for treating an individual having an abnormal boneor a dysplasic bone.
 3. The method according to claim 1, for treating anindividual having an abnormal bone by increasing the size of a bonegrowth plate in said abnormal bone.
 4. The method according to claim 1,for treating an individual having a dysplasic bone.
 5. The methodaccording to claim 1, wherein the FGF-9 variant comprises the amino acidsequence set forth in SEQ ID NO: 11 or
 13. 6. The method according toclaim 1, wherein the FGF-9 variant comprises the amino acid sequence setforth in SEQ ID NOS: 14 or
 15. 7. The method according to claim 1,wherein the FGF-9 variant is linked to a bioactive agent.
 8. The methodaccording to claim 7, wherein the bioactive agent is CNP(1-22).
 9. Themethod according to claim 8, wherein the FGF-9 variant comprises theamino acid sequence set forth in SEQ ID NOS: 16 or
 17. 10. The methodaccording to claim 1, wherein the method is used for elongating saidabnormal bone.
 11. The method according to claim 1, wherein the FGF-9variant is an amino acid sequence selected from the group consisting ofSEQ ID NOs: 11, 13, 14, 15, 16 and 17.